MOLECULAR
PHYLOGENETICS
AND
EVOLUTION
Vol. 1, No. 1, March, pp. 17-25, 1992
Phylogenetic Relationships among Middle American Pocket Gophers (Genus Orthogeomys) Based on Mitochondrial DNA Sequences PHILIP
D. SUDMAN AND MARK 5. HAFNER
Museum of Natural Science and Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803-32 76 Received August 23, 1991; revised February 3, 1992
interspecific interactions, phylogenetic relationships, or even the basic biology of these rodents. The fossil record of Orthogeomys is meager, with only a single late Pleistocene specimen known [O. onerosus (Russell, 1960, 1968)l. In the absence of fossil data from which to reconstruct the phylogenetic history of the Orthogeomys lineage, relationships among the extant taxa have been inferred from genetic data. Hafner (1982) demonstrated that the three subgenera are well differentiated from each other based on both protein-electrophoretic and immunological evidence. However, the relationships among these taxa remain unclear. Phenetic analyses of electrophoretic data suggest that the subgenera Orthogeomys and Heterogeomys are sister taxa, whereas cladistic (locus-by-locus) analyses and immunological data indicate that Mucrogeomys is more closely related to Heterogeomys (Hafner, 1979, 1982). Relationships among Orthogeomys at the species level also are unresolved, especially within the subgenus Macrogeomys (Hafner, 1991); each of the other subgenera contains only two species. Protein-electrophoretic data are strongly indicative of a sister&axon relationship between 0. cherriei and 0. heterodus and between 0. cauator and 0. duriensis. However, the placement of 0. underwoodi within Macrogeomys could not be resolved definitively (Hafner, 1991). Cladistic analysis placed 0. underwoodi as a sister taxon to the cherriei-heterodus lineage, whereas parsimony analyses of the same data based on the presence-absence of alleles ally 0. underwoodi with the cavator-dariensis clade. The sixth species within this subgenus, 0. matugulpae, was not available for analysis, and the placement of a newly described member of the subgenus, 0. thueleri (Alberico, 1990), has yet to be determined. In an effort to better understand the relationships within and among the subgenera of Orthogeomys, we used nucleic acid sequence data from two regions of the mitochondrial genome for phylogenetic analyses. These results are compared with those of similar stud-
Relationships among members representing each of the three subgenera of the Middle American rodent genus Orthogeomys (Rodentia: Geomyidae) were studied by comparing DNA sequence data from two regions of the mitochondrial genome. Results from 527 bp from the 16 S rDNA region and a 402.bp fragment of the cytochrome b gene indicate that the three subgenera are well differentiated genetically, with the subgenus Orthogeomys being distantly related to Macrogeomys and Heterogeomys, and Macrogeomys appearing as the most derived. Within the subgenus Macrogeomys, 0. he&o&s and 0. cherriei form a distinct clade, as do 0. dariensis and 0. cavator. As with previous proteinelectrophoretic studies, the placement of 0. underwood could not be determined definitively within the subgenus Macrogeomys. We interpret our inability to determine phylogenetic relationships among these three clades as evidence for a rapid phyletic radiation within this subgenus. Sequence divergence estimates indicate that the Macrogeomys radiation took place following the time of completion of the Panamanian land bridge (1.9-2.9 myal. Additionally, the near identity of sequences of a newly described species, 0. thaeleri, with those of 0. dariensis (percentage sequence divergence = 0.3%) suggests that the two may be conspecific. B 1X42 Academic Prees, Inc.
INTRODUCTION
Middle
American
pocket gophers of the genus Ormembers of the rodent family Geomyidae. The genus, which ranges from Mexico south to northwestern Colombia and occupies a wide range of habitats, comprises three subgenera, Orthogeomys, Heterogeomys, and Macrogeomys. Because of their subterranean habits and the relative inaccessibility of major portions of their geographic ranges, very little is known about the distributions, thogeomys are among the least-studied
17 1055-7903/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
18
SUDMAN
AND
ies based on protein electrophoresis to elucidate degrees of divergence, patterns of descent, congruence of evolution in different (independent) genomic regions, and specific status of the taxa in question. MATERIALS
AND
METHODS
Total nucleic acid preparations were obtained from liver or kidney following the phenol/chloroform extraction techniques summarized by Hillis et al. (1990). Amplifications of specific regions of the mitochondrial DNA (mtDNA) genome were then performed via the polymerase chain reaction (PCR) using the following sets of primers: 16 S rDNA, L2510 (5’-GGAATTCCCGCCTGTTTATCAAAAACAT-3’) and H3062 (5’~GGAATTCCCTCCGGTTTGAACTCAGATC-3’); cytochrome b, L14724 (5’-CGAAGCTTGATATGAAAAACCATCGTTG-3’) and H15149 (5’-CCTCAGAATGATATTTGTCCTCA-3’). L and H refer to the 3’ position of the primers in relation to human mtDNA light and heavy strands, respectively (Anderson et al. 1981). Double-stranded PCR amplifications using Thermus acquaticus DNA polymerase (Saiki et al., 1986, 1988) were performed in 50 or loo-k1 total reaction volumes following the balanced primer procedure described by Allard et al. (1991). Twenty-five to 35 cycles were performed with the following cycling parameters: 16 S rDNA-1 min denaturation at 93°C 1 min annealing at 48”C, and 1 min extension at 72°C; cytochrome b-l min denaturation at 93”C, 1 min annealing at 56”C, and 1 min extension at 72°C. Five microliters of the double-stranded product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized under UV to assess reaction success. Five microliters of the double-stranded product was then used to generate single-stranded DNA in loo-k1 reactions following the same procedures outlined above, except that only one primer was added to the reaction mixture. Single-stranded DNA was generated for both the heavy and the light mtDNA strands, and the products were cleaned by multiple washings with H,O through Ultrafree-MC 30,000 NMWL filters (Millipore Corp., Bedford, MA) and concentrated to a final volume of 50 p-1. Seven microliters of the cleaned single-stranded templates was then used for DNA sequencing using T7 DNA polymerase (Sequenase version 2.0, United States Biochemical, Cleveland, OH) following standard dideoxy chain termination protocols (Sanger, 1977). Both mtDNA strands were sequenced to within approximately 30 bp of the sequencing primer. Sequences were aligned using the ALIGN software program (version 1.0, Scientific & Educational Software, 1989) and trees were constructed using PAUP, version 3.0 (Swofford, 1989) and PHYLIP, version 3.3 (Felsenstein, 1990). Because of uncertainty about the
HAFNER
relationships among genera within the tribe Geomyini (Hafner, 1982; Honeycutt and Williams, 1982), Thomomys talpoides (tribe Thomomyini) was used as an outgroup for all analyses. Tree-building procedures included maximum parsimony (Fitch, 1971), maximumlikelihood (Felsenstein, 1981), and Fitch and Margoliash (1967) methods for fitting trees to distance matrices generated following the assumptions of the Kimura two-parameter model (Kimura, 1980). Analyses were performed for the entire data set as well as for transversions only. Additionally, Mantel’s test (Mantel, 1967) was used to compare a matrix of genetic distance values from protein data (Hafner, 1991) with the percentage sequence divergence estimates generated in this study to determine the relationship between nuclear and mitochondrial data sets. The following specimens were examined: Orthogeomys hispidus-Louisiana State University Museum of Natural Science (LSUMZ) 29232 0 ; Belize: Cayo District, 3 km W Belmopan. Orthogeomys thaeleri-LSUMZ 30057 0 ; Colombia: Departamento de1 Chock, Ensenada de Utria, Parque National Natural Utria, Municipio de Bahia Solano, -10 m. Orthogeomys cavator-LSUMZ 29490 8 ; Costa Rica: Prov. San Jose, 3 km SW Division, 2200 m. Orthogeomys cherriei-LSUMZ 29539 6 ; Costa Rica: Prov. Guanacaste, 2 km E Tronadora. Orthogeomys heterodus-LSUMZ 29498 0 ; Costa Rica: Prov. Cartago, 2 km W Santa Rosa, 2300 m. Orthogeomys underwoodi-LSUMZ 29537 9 ; Costa Rica: Prov. Alajuela, 3 km S Orotina. Orthogeomys grandis-Museum of Vertebrate Zoology (MVZ) 154079 c?; Mexico: Michoacan, 3.6 km (by road) N Arteaga. Orthogeomys dariensis-LSUMZ 25436 d ; Panama: Prov. Darien, 6 km SW Cana, 1200 m. Thomomys talpoides-LSUMZ 29301 Q; New Mexico: Sandoval Co., 3 km N, 17.8 km E Jemez Springs. RESULTS
Alignment of the sequences resulted in a final data set of 929 bp (Fig. l), including 527 bp from 16 S rDNA [homologous to the region between human positions 2550 and 3062 (Anderson et al., 198l)l and 402 bp from the cytochrome b gene coding region [human positions 14,747 to 15,149 (Anderson et al., 1981)]. The sequences from 0. duriensis and 0. thaeleri were identical, with the exception of three transitions, one within the cytochrome b sequence and two within the 16 S rDNA sequence (see asterisks, Fig. 1); 0. thaeleri was therefore omitted from all further analyses. The majority of variation within the 16 S rDNA was partitioned within presumed hypervariable regions, whereas variation within the portion of the cytochrome
PHYLOGENETIC
RELATIONSHIPS
AMONG
19
Orthogeomys
>>16S rDNA AGTGACAAAA GTTAAACGGC CGCGGTATTC TGACCGTGCA AAGGTAGCAT AATCATTTGT . .. .. . . .......... .......... .......... .......... .......... .. ... . . .......... .......... .......... .......... .......... . .. .. . . .......... .......... .......... .......... .......... .......... . .. .. . . .......... .......... .......... .......... . . ... . . .......... .......... .......... .......... .......... .. ... . . .......... .......... .......... .......... .......... .. ... . . .......... .......... .......... .......... .......... .. ... . . .......... ........ c. .......... .......... .T...C ....
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
0. 0. 0. 0, 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
TCTCTAATTA AGGACTTGTA TGAATGGCAC GACGAGGGTT .......... .. A ........... C ............... .......... ...... C ........... G. A....A .... .......... .............................. .......... .............................. .......... .............. C ............... .......... .............. C ............... .......... .............. C ............... . . . T . . . . . . .................. GT ..........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
TCTATGAAAT TGACCTTTCC GTGAAGAGGC GGAAATAAAT AAATAAGACG .......... .......... .......... .......... .......... . . A ....... .... T ..... .......... .......... T ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TTCA T .........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
ATGGAGCTTC .......... . . . . . . . . .T .......... .......... .......... .......... .......... . . . . . . . . .A
AATTAAA-T.......... .G....T... .......... .......... .......... . . . . .T . . . . . . . . .T . . . . . . . . . . TA.A
ACTTTATAAT . . . . . . . . .C . ..C....C. . . . . . . . . .C . . . . . . . . .C . . . . . . . . .C . . . . . . . . .C . . . . . . . ..c G..C.TCTTA
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
AATAACTAAC ......... T ...... ..-. ......... T ......... T ......... T ......... T ......... T ... ..- ... A
TTAA-AAGT .......... ...... G ... .......... .......... .. T ....... .......... .......... -G..AAG ...
AAAAAA-TTT TGGTTGGGGT GACCTCGGAG CACAACAAAA ........................................ T ... ..AC ...................... ..T.G ..... ........................................ ........................................ .................................. G ..... ....................... . ........ ..G.G ... ................................ ..G..C .. T .. ..- ........................ T.T.GT . ..C
TTACTGTCTC TTACATTCAA ............. T ... T .. .A .................. ............. T ... T .. ............. T ... T .. ............. T ... T .. ............. T ... T .. ............. T ... T .. .A ........ .C.T...TT. AGAAGACCCT ...... .. ...... .. ...... .. ...... .. ...... .. ...... .. ...... .. ...... ..
* TTTAAATATT . . . . . ..TCA C.....A.CA . . . . . . . . CA . . . . . . . . CA C.......CA . . . . . . c.AA c.....c.AA .GA.TT..AA
ACTCCA-CAG . . ..T....A ..CTA..... . . ..T....A . . ..T.... G .......... . . . . . . ...* .......... .A..A.AT.A
GAA-ATAACA .......... . ..T.C.... .......... .......... . . ..G..... I......... .......... ..G.......
FIG. 1. Aligned sequences for each of the nine species of pocket gophers (Orthogeomys and Thomornys) analyzed. Dots indicate identity with the 0. h&i&s sequence as depicted in the first row, and dashes indicate gaps inserted to maximize homologies. Asterisks designate the three positions where 0. dariensis and 0. thaeleri differ in sequence.
b gene was evenly dispersed throughout. Within Orthogeomys, 65 of the 527 16 S rDNA positions were variable; 67.2% of these differences were transitions, and 32.8% were transversions. Within the cytochrome b region, 105 of 402 sites were variable within Orthogeomys (73.3% transitions and 26.7% transversions), resulting in amino acid changes at 13 of the 134 codons. The distribution of the variable cytochrome b sites with respect to first,
second, and third codon positions was 20.0, 4.8, and 75.2%, respectively. Percentages of silent and replacement transitions and transversions for each position are given in Table 1. The 0. underwoodi sequence was unique in possessing a 3-bp deletion at the third or fourth amino acid position. Although deletions have not been previously reported in this region of the cytochrome b genome (Irwin et al., 1991; Howell, 1989; Smith and Patton, 19911, codon deletions and additions
20
SUDMAN
AND
HAFNER
* 0. 0. 0. 0. 0. 0, 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
CCTCCGAAAG ACTATTATT- TAGACCTGCA AGTCAACATT AG..A. C ...... A ............ .............. .............. A . ..A T ....... A ............ .......... .T..AA..A. ....... A ...... G.T ... .......... .T..AA..A. C ...... A ...... G.T ... A ............ ............ ..AG..C. ....... .............. AG .... .. ..A.AA ............ .............. AG .... .... A.AA ............ .............. AC .... ...... CA ...... ..T ...
-AACATAA-G ... T ..... A ... T ...... ... T.C .. .A . ..T.C . ..A ... T ..... A ... T ..... A ... T ..... A TT.T-...A.
T-CCT-AATT ... AC ..... C..T ...... .T.A ...... .T.A ...... ... AC ..... ... AC ..... ... AC ..... . ..T.A ....
TAACAGCGCA ATCTTATTGG
0.
cavator
0. 0. 0. 0, 0. 0. T.
grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
GACCCAGAAA TCTGATCAAT GGACCAAGTT ACCCTAGGGA .................... .A .................. .................... .................... .................... .A .................. .................... .A .................. .................... .A .................. .................... .A .................. .................... .A .................. .. T ....... C ......... ....................
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
AGAGTCCATA TCGATAATAA GGTTTACGTA CTCGATGTTG GATCAGGACA TCCTAATGGT ............................ AC .................... ...... C ... ............................ AC .................... .......... ............................ AC .................... .......... .......................... ..A C .................... .......... ............................ AC .................... .......... ................... G ...... ..A C .................... .......... .......................... ..A T .................... .......... .......... ..... T ................ C ..... AC ....................
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
GCAGAAGCTA .......... .......... .......... .......... .......... .......... .......... ..........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
CTTTTAGAGT GGGTGATTAT ....... G .. A..C..A ... ....... T ..... AA ..... ....... G .. A..C..A ... ....... G .. A..C..A ... ....... G ........ C ... ....... G .. A. ........ ....... G .. A ......... .A...T.T ... ..GA..A .A
0. hispidus
TTAAGGGTTC .......... .... T ..... .......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... .......... .......... ..........
16s I-DNA cytochrome h GTTTGTTCAA CGATTAAAGT CCTACGTTAC TGTTAATATG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..T.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GG.. . ..T
................................. ...................................
.... T ....
T---. ................................... ............................ G. ... ..C .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. . . . . . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . .G. . . .
TTTTAACAGT .......... .......... .......... .......... .......... ........ A. .......... ..........
TGGTACGAM GTAACTGGAT GGTTGTGGTG A .......... ....... G .. ......... .......... ... G .............. G. .......... ... G ................ .......... ... G ................ .......... ... G .............. G. ....... G .. ... G ... A ............ .......... .C.G ................ .. ..G..T .. A ......... ..A..G ....
FIG. l-Continued
are known to occur within the cytochrome b coding region in other mammals (Irwin et al., 1991). Uncertainty about the exact position of the deletion exists because both the third and the fourth amino acid codons are identical in three of the Orthogeomys species examined. That this deletion is widespread was confirmed by examining the sequences of three additional specimens of 0. underwoodi from throughout its range. All three specimens contained identical sequences within the region in question. Regardless of which tree-building algorithm was
used, the cytochrome b data set yielded a single tree with the same topology as that shown in Fig. 2a. Independent analysis of the 16 S rDNA data yielded three trees, all, of which were identical to the cytochrome b tree except for the replacement of 0. underwoodi within the subgenus Mucrogeomys. Because of this overall similarity, the cytochrome b and 16 S rDNA data sets were combined for all remaining analyses. Percentage sequence divergence within Orthogeomys ranged from 2.77 to 13.56, with 0. grandis showing the greatest degree of divergence from all other
PHYLOGENETIC
RELATIONSHIPS
TCCAACTACT ..C ... ..C GT ........... G..T ...... G..T ...... T ...... ..T ...... ..T ...... ..T.A .......
AMONG
TTAAAGCCGA ..G..A .... ..A..A. ..G..A .... ..G..A .... ..G..A .... ..G..A..A. ..G..A..A. ..A..C.
21
Orthogeomys
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
GATTATAGAG .T..G ...... .... G ..... .G..G..A .. .G..G..A .. .... G ........ .G..G ...... .G..G ...... .... G ......
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus oherriei talpoides
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
AGAGATGTCC GGATAAAGAT CGTTATGTGA TGTGTAGACT GTGTGAATGT CGGAAGAGTA .A..T ........... ..A ............ .A.....T ............ ..A.....C. . ..AT ..... .A....GA ................. ..T ... ..G..G ... ..A ....... .... T ............ A ............ .A..C..T ............ ..A.....C. .... T ............ A ............ .A..C..T ............ ..A.....C. .A..T .......... ..A .. ..A ............ ..T .. A ...... ..A ..A ....... .A..T .......... ..A .............. ..C..T ............ ..A ....... .A..T .......... ..A .............. ..A ....... ..C..T ............ ... AT..G .. T.....G ... ..A.....A. ..... ..T ... ..G ...... ..A..A .... * GACATCGTGT GTAAACGGCA CTGCATTTGA TGCCGACTAA TTAGGCTATG TATGTACGGT .T ................. . ............ ..T .............. . .......... .T...T .... A ...... ..T ..A ......... ..T ...... ..A ................ .T...T ............. . ......... . .. ..T .............. . G ......... .T...T .... A ...... ..G ......... . .. ..T .............. . G ......... .T ........ A ...... ..G ......... . .............................. . .......... .T ................. . ............................. . .......... .T ........................... . A .................. .T..AT.A .. A ...... ..T ..A ....... ..A ... ..A ...... ..A. .A .........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
TGCCGCGGAG GGATAAGAAG .A..A ..... A ......... .A.....A ............ .A..A ............... .A..A ............... .A..A ..... .A ........ .... A ............... .A..A ............... .... T ..... A ........ A
TAAACGGAAA .......... . . G . . . . . G. .......... .......... .......... . . . . . . . . G. .*... A . . . . . . . . ..A.T.
TATAAGTGTA .G........ .G..G..A.. .G........ .G........ .G........ .G........ .G........ .G..G..A..
GCCTGCTCCT .................. ......... A .................. .................. ......... C ......... G ......... G A ..............
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
CTAGGATAGA . . . .A . . . . . . . . . A . . . A. . . . .A. . . . . . . . .A . . . . . . . . .A . . . . . . . . . . 0. G . . .C.....G.. . . . . . . . G. .
AATATGTCTT .......... T..GA..... .......... . ..*...... G..G.T.... G . . . . T.... . . . . .T . . . . . . . . .T . . . .
TGTACTTTGT ........ A. .......... ... ..C..A. ... ..C..A. ........ A. ........ A. ........ A. ........ A.
AGCCGTAAAA ........... .A..T..GG. .T ........ .T ........ .......... ........... .A ............ .A.....G ..
TAATGATAAA AATTGATATC ..CA .. ..G .......... CG..A .. ..G G .......... .G.C ... ..G .......... .G.C ... ..G .......... .G ..... ..G G....G .... ..C ................ ..C ............. .G ........... ..T ....
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
GGTGACGTAA .......... .T........ .......... . ...*..... . . . . . . .C. . .......... .......... .A..T..G..
GCATCCTATA CT........ A..C..A..G .T........ .T........ .T........ .T........ .T.......G
CATGATGGT . . . ..C... ......... ..c..c... ..C..C... ......... . . . ..C... . . . . .C. . .
A.....A...
..GA.....
FIG. l-Continued
GGGATAATCC . . . . . G. . . . .......... . . . . . G. . . . . . . . . G. . . . . . . . . G. . . . . . ..CG.... . . . . . G. . . . . . ..CG.A..
TGATACGAAT .......... .... ..AG .. .......... .......... .......... .......... .......... .T ........
TAAGATGTTT ... A ...... .GG..A .... . .GA ...... ..GA ...... ... A ...... ... A ...... ... A ...... ..........
TAAATGATAC G. .. G ....... G. G. .......... .......... .......... A ....
22
SUDMAN
TABLE
AND HAFNER
1
Distribution of Silent and Replacement Nucleotide Changes for Pairwise Comparisons between Seven Species of Orthogeomys at 402 Sites within the Cytochrome b Gene Codon position
Transitions Silent Replacement Transversions Silent Replacement
First
Second
Third
95 (10.6) 49 (5.5)
0 (0.0) 18 (2.0)
499 65.8) 10 (1.1)
5 (0.6)
0 (0.0)
12 (1.3)
18 (2.0)
8.)
(19.1) 18 (2.0)
170
Note. Percentages are given in parentheses.
taxa (Table 2). Based on sequence divergence alone, the subgenus Orthogeomys (represented by 0. grandis) is the most divergent. The subgenera Macrogeomys and Heterogeomys (represented by 0. hispidus) display a moderate degree of similarity (average percentage sequence divergence = 7.26). Within the subgenus Macrogeomys, sequence divergence ranges from 2.77 between 0. cherriei and 0. heterodus to 5.92 between 0. cherriei and 0. dariensis. Results of the Mantel’s test comparing the percentage sequence divergence values (this study) with Nei’s (1978) unbiased genetic distance (Table 2) computed from the protein electrophoretic data of Hafner (1991), indicated a high level of concordance between the two matrices (t = 2.15, zvO.05). In general, transition-to-transversion ratios (Table 3) decreased with increasing sequence divergence. Within the subgenus Macrogeomys, ratios ranged from 2.00 to 5.25, whereas ratios among the three subgenera of Orthogeomys ranged from 2.00 to 2.88. Transitionto-transversion ratios were lowest for comparisons between Orthogeomys and Thomomys (ranging from 1.03 to 1.28), supporting the general assumption that transitions saturate over time (Brown, 1982). All three tree-building procedures resulted in the same interpretation of the relationships among the three subgenera of Orthogeomys, regardless of whether they were based on the entire data set or transversions only. Based on these analyses, the subgenera Heterogeomys and Macrogeomys are sister taxa within the genus Orthogeomys, and show a closer association to each other than either does to the subgenus Orthogeomys. Members of the subgenus Macrogeomys show a high degree of overall similarity. The position of 0. underwoodi within the subgenus Macrogeomys differed depending on the method of analysis. In all of the analyses, 0. cherriei and 0. h&rodus were depicted as sister taxa, as were 0. dariensis and 0. cavator. 0. underwoodi was portrayed as the
I
13 ati 44
T.ta#waea
b.)
FIG. 2. Phylograms resulting from parsimony analyses of mtDNA data depicting relationships within the genus Orthogeomys. Thomomys tulpoides was included as an outgroup. The numbers on each branch indicate branch lengths. (a) The most-parsimonious tree based on analyses of all mutations. The consistency index (CI) for the tree is 0.825. Although this topology was supported by maximum-likelihood analysis, a Fitch-Margoliash analysis of distance values placed 0. underwoodi as a sister taxon to the 0. chrriei-0. heterodus clade as in Fig. lb. (b) Most-parsimonious tree when only transverions were used in the analyses (CI = 0.896). The topology of this tree was supported by all three tree-building algorithms.
outgroup of all other Macrogeomys in both maximum likelihood and parsimony analyses (Fig. 2a), but was placed as a sister group to the cherriei-heterodus clade in the Fitch-Margoliash analysis of distance values. It should be noted, however, that a parsimony tree containing the same topology as that obtained from the distance analysis (i.e., 0. underwoodi allied with the cherriei-heterodus clade) was only three steps longer than the most-parsimonious tree (CI = 0.817), and one depicting 0. underwoodi as the sister taxon to the cauator-dariensis clade was only two steps longer (CI = 0.820). Trees based on transversion data alone were more consistent than those derived from both transitions and transversions (Fig. 2b). All three tree-building algorithms were in agreement in the placement of 0. underwoodi as the sister group to 0. cherriei and 0. heterodus. However, a parsimony tree one step longer than that illustrated in Fig. 2b depicted the relation-
PHYLOGENETIC
RELATIONSHIPS
TABLE
AMONG
23
Orthogeomys
2
Percentage Sequence Divergence (Rimura, 1980) for Orthogeomys Taxa and the Outgroup, Thomomys, Derived from Comparison of 929 bp from Two mtDNA Regions (above Diagonal) and Nei’s Unbiased Genetic Distance (Nei, 1978) for the Same Taxa Computed from the Data of Hafner (1991) (below Diagonal) 1 1 2 3 4 5 6 7 8
0. 0. 0. 0. 0. 0. 0. T.
gmndis hispidus cavator cherriei dariensis heterodus underwoodi tulpoides
Note.
Thomomys
0.693 1.135 0.921 1.217 0.937 0.992 talpoides
was not
2
3
4
11.39 1.067 0.804 0.958 0.947 0.837 -
13.17 7.23 0.195 0.256 0.229 0.193 -
13.48 7.25 5.81 0.305 0.093 0.092 -
included
in the study
by Hafner
ships within the subgenus Mucrogeomys as an unresolved trichotomy between 0. underwoodi and the cavator-dariensis and cherriei-heterodus clades (CI = 0.888).
DISCUSSION In his revision of the subfamily Geomyinae, Russell (1968) synonymized the genera Orthogeomys, Heterogeomys, and Macrogeomys into the genus Orthogeomys. Although he retained each of the former genera as subgenera, Russell stated: “A revision of the genus is needed; it might show that the currently recognized subgenera are artificial, and that a different arrangement of the species would more clearly express their evolutionary relationships” (Russell, 1968, p. 510). Based on the sequence data presented herein and the protein data of Hafner (1982), each of the subgenera appears to be genetically well differentiated, at least within the context of the species examined. Although the relationships among the subgenera were not readily apparent from previous work based on protein data, mtDNA sequence data indicate that Mucrogeomys and Heterogeomys are more closely related to each other than either is to Orthogeomys. TABLE
1 2 3 4 5 6 7 8
0. 0. 0. 0. 0. 0. 0. T.
gmndis hispidus cavator cherriei dariensis heterodus underwoodi talpoides
Note.
Transitions/trausversions
Pairwise
Comparisons 929.bp
1
2
2.00 2.44 2.03 2.62 2.42 2.00 1.28
64132 2.50 2.00 2.88 2.59 2.50 1.03 are
shown
78132 45118 2.00 3.57 2.38 2.36 1.08 the
diagonal
13.56 6.99 4.97 2.77 5.67 0.143 -
7
8
11.82 7.26 5.34 4.75 5.69 5.10 -
16.30 15.95 17.21 16.92 16.92 16.05 16.81 -
(1991).
Interspecific relationships within the subgenus Mczcas determined by protein electrophoresis (Hafner, 1991) are well supported by all analyses of the mtDNA data. 0. cherriei and 0. heterodus are characterized as being the least differentiated from each other in terms of their percentage sequence divergence and absolute numbers of transitions and transversions. 0. cavator and 0. dariensis also display close affinities at all levels, with slightly higher percentage sequence divergence and numbers of transitions and transversions than the cherriei-heterodus clade. As with analyses of protein data, the placement of 0. underwoodi within the subgenus Mucrogeomys could not be determined definitively. The phylogenetic placement of 0. underwoodi within Macrogeomys therefore remains unresolved, and our present understanding of the relationships within the subgenus can best be represented as an unresolved trichotomy involving 0. underwoodi and the cavator-dariensis and cherriei-heterodus lineages. It could be argued that our inability to resolve completely evolutionary relationships within the subgenus Mucrogeomys is simply the result of an insufficient number of informative sites in the mtDNA data set. It seems more likely, however, that the lack of resolution rogeomys
3 Based
on the
4
5
6
75137 42121 34117
76129 49117 2517 36/16
80133 44117 31113 2114 38112 3.09 1.11
2.25 5.25 2.82 1.08 and
6
12.50 7.58 3.57 5.92 0.378 0.272 -
of Transitions and Transversions mtDNA Sequences Reported Herein 3
above
5
transition-to-transversion
3.17 2.85 1.08 ratios
below
7 66133 45118 33114 31111 37113 34111 1.16 the
diagonal.
8 74158 66164 71166 70165 70165 68/61 72162 -
24
SUDMAN
is actually evidence for a rapid phyletic radiation within the subgenus. Kraus and Miyamoto (1991) suggest that short internode lengths and near identical levels of sequence divergence among taxa are consistent with hypotheses of rapid origin and radiation of lineages. As can be seen in the parsimony trees (Figs. 2a and 2b), the internode lengths between the three lineages of Macrogeomys are relatively short compared to the terminal branches. The other two tree-building algorithms used in this study support these observations; in each case, internodal lengths between the lineages of Macrogeomys are small compared to terminal branch lengths. Estimates of sequence divergence between 0. underwoodi and the other species of Macrogeomys (Table 2) are similar, as are the absolute numbers of transversions (Table 3). Together this evidence seems to point toward a rapid radiation involving the ancestors of each of the cauator-dariensis, cherriei-heterodus, and 0. underwoodi lineages. In an effort to resolve more clearly the relationships within Macrogeomys, we used a jackknifing procedure (Lanyon, 1985) with each of the tree-building algorithms. Lanyon (1985) advocated use of a jackknife approach as a means of identifying internal inconsistencies within a data set. In each case, jackknifing indicated that relationships within the subgenus Macrogeomys were best represented as an unresolved trichotomy between 0. underwoodi and the cauatordariensis and cherriei-heterodus clades. Studies based on protein-electrophoretic data also failed to resolve completely the relationships between 0. underwoodi and the other members of the subgenus (Hafner, 1991). Thus, studies of the nuclear genome (Hafner, 1991) are concordant with studies of the mitochondrial genome (this study) and, together, they suggest a rapid phyletic radiation early in the history of the subgenus Mucrogeomys, because both nuclear and mitochondrial DNA data sets failed to resolve unequivocally relationships within the subgenus. Brown et al. (1979) and Shields and Wilson (1987) have suggested that vertebrate mtDNA evolves at a rate of approximately 2% per million years. Using this as a rough estimate of divergence rates, we calculated times since divergence based on our data (Table 2). Estimates of time since divergence between Orthogeomys and Thomomys places the cladogenic event between the tribes Geomyini and Thomomyini at approximately 8 million years ago (mya). This is in close agreement with divergence times estimated from the fossil record, which indicates that Thomomys split from the main geomyid lineage during the late Miocene (Russell, 1968). Within the genus Orthogeomys, sequence data indicate that the subgenus Orthogeomys diverged from the Macrogeomys-Heterogeomys lineage approximately 6 mya, and that Macrogeomys and Heterogeomys diverged from each other approximately 3.6 mya. The
AND
HAFNER
first of these estimates is in close agreement with Russell’s (1968) proposed timing of the major radiation within the tribe Geomyini during the mid Pliocene. Our estimate of time since divergence of Heterogeomys and Macrogeomys (3.6 mya) places this event contemporaneous with the completion of the Panamanian land bridge (Marshall et al., 1979). Considering that the present distribution of Mucrogeomys straddles the Panamanian isthmus, it seems reasonable that the principal phyletic radiation of this group was triggered by major geologic events in the region, including the availability of new habitat resulting from geologic uplift. Divergence estimates within the subgenus Macrogeomys are on the order of 1.9 to 2.9 mya. Although these time estimates are considerably more recent than those estimated by Hafner (1991) based on Nei’s genetic distances (4.5 mya), they are well within the proposed historical biogeographic framework proposed for the subgenus (Hafner, 1991). Hafner (1991) speculated that the current distributions and relationships within the subgenus may be directly related to isolating effects of repeated glacial and interglacial periods of the Quaternary. These events would have essentially transformed the mountainous regions of Middle America into a series of islands, resulting in repeated fragmentation, and perhaps speciation, of ancestral populations. Divergence estimates roughly corresponding to the early Pleistocene (this study) are consistent with this hypothesis. 0. thaeleri, originally described as distinct from 0. dariensis based on pelage and cranial differences (Alberico, 19901, occupies a small range in the northwestern corner of Colombia within 50 km of the known distribution of 0. dariensis. Pocket gophers have been shown to exhibit a high degree of morphological variation over short geographic distances due to local habitat differences (Patton and Brylski, 19871. It is possible, therefore, that populations representative of 0. dariensis and 0. thaeleri are distinct morphologically, but are, nevertheless, members of a single gene pool. 0. thaeleri and 0. dariensis had an estimated sequence divergence of only 0.3%, whereas all other intrageneric comparisons exceeded 2.7%. Irwin et al. (1991) reported intraspecific sequence variation of 0 and 1.7% for cow @OS tuurus) and long-beaked dolphin (SteneZZa Zongirostris), respectively, over the entire cytochrome b gene. Smith and Patton (1991) reported that sequence differences among conspecific populations of the rodent genus.Akodon ranged from 0.25 to 8% based on the same portion of the cytochrome b gene examined in this study. Although it is certainly not valid to make taxonomic decisions based solely on percentage sequence divergence, we feel that because the estimate of mtDNA sequence divergence between 0. thaeleri and 0. dariensis falls well within the range of values reported for conspecific populations of other mammals,
PHYLOGENETIC
RELATIONSHIPS
Q. thaeleri may represent a geographic variant of 0. duriensis. However, additional sampling from the region between the two forms is needed before formal taxonomic changes can be considered. ACKNOWLEDGMENTS We thank J. Spatafora for technical assistance with PCR and sequencing protocols, J. Derr and S. Davis for the 16 S rDNA primers, M. Alberico for tissue samples from 0. thueleri, D. J. Hafner for specimen collection, and J. Demastes, D. Good, and R. Zink for helpful comments and criticisms of earlier drafts of the manuscript. This research was supported by National Science Foundation Grant BSR-8817329.
REFERENCES Alberico, M. (1990). A new species of pocket gopher (Rodentia: Geomyidae) from South America and its biogeographic significance. In “Vertebrates in the Tropics” (G. Peters and R. Hutterer, Eds.), pp. 103-111, Museum Alexander Koenig, Bonn. Allard, M. W., Ellsworth, D. L., and Honeycutt, R. L. (1991). The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction. BioTechniques 10: 24-26. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. P., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R., and Young, I. G. (19811. Sequence and organization of the human mitochondrial genome. Nature 290: 457-465. Brown, W. M., George, Jr., M., and Wilson, A. C. (1979). Rapid evolution of animal mitochondrial DNA. Proc. N&Z. Acad. Sci. USA 76: 1967-1971. Brown, W. M., Prager, E. M., Wang, A., and Wilson, A. C. (1982). Mitochondrial DNA sequences of primates: Tempo and mode of evolution. J. Mol. Evol. 1% 225-239. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: A maximum likelihood approach. J. Mol. Evol. 17: 368-376. Felsenstein, J. (1990). “PHYLIP 3.3 Manual,” University of California Herbarium, Berkeley. Fitch, W. M. (1971). Toward defining the course of evolution: Minimum change for a specified tree topology. Syst. 2001.20: 406-416. Fitch, W. M., and Margoliash, E. (1967). Construction of phylogenetic trees. Science 155: 279-284. Hafner, M. S. (1979). “Evolutionary Relationships and Systematics of the Geomyidae (Mammalia: Rodentia).” Unpublished Ph.D. dissertation, University of California, Berkeley. Hafner, M. S. (1982). A biochemical investigation of geomyoid systematics (Mammalia: Rodentia). 2. Zool. Syst. Evolutions forsch. 20: 118-130. Hafner, M. S. (1991). Evolutionary genetics and zoogeography of Middle American pocket gophers, genus Orthogeomys. J. Mammal. 72: l-10. Hillis, D. M., Larson, A., Davis, S. K., and Zimmer, E. A. (1990). Nucleic acids III: Sequencing. In “Molecular Systematics” (D. M.
AMONG
Orthogeomys
25
Hillis and C. Moritz, Eds.), pp. 318-370, Sinauer Associates, Sunderland, MA. Honeycutt, R. L., and Williams, S. L. (1982). Genie differentiation in pocket gophers of the genus Pappogeomys, with comments on intergeneric relationships in the subfamily Geomyinae. J. Mammal. 63: 208-217. Howell, N. (1989). Evolutionary conservation of protein regions in the proton-motive cytochrome b and their possible roles in redox catalysis. J. Mol. Evol. 29: 157-169. Irwin, D. M., Kocher, T. D., and Wilson, A. C. (1991). Evolution of the cytochrome b gene in mammals. J. Mol. Evol. 32: 128-144. Kimura, M. (1980). A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120. Kraus, F., and Miyamote, M. M. (19911. Rapid cladogenesis among the Pecoran ruminants: Evidence from mitochondrial DNA sequences. Syst. Zool. 40: 117-130. Lanyon, S. M. (19851. Detecting internal inconsistencies in distance data. Syst. Zool. 34: 397-403. Mantel, N. (1967). The detection of disease clustering and a general regression approach. Cancer Res. 27: 209-220. Marshall, L. G., Butler, R. F., Drake, R. E., Curtis, G. H., and Tedford, R. H. (1979). Calibration of the great American interchange. Science
204:
272-279.
Nei, M. (1978). Estimation distance from a small
of average heterozygosity and genetic number of individuals. Genetics 89:
583-590.
Patton, J. L., and Brylski, P. V. (1987). Pocket gophers in alfalfa fields: Causes and consequences of habitat-related body size variation. Am. Nat. 130: 493-506. Russell, R. J. (1960). Pleistocene pocket gophers from San Josecito Cave, Nuevo Lebn, Mexico. Univ. Kans. Pub., Mus. Nat. Hist. 9: 539-548.
Russell, R. J. (1968). Evolution and classification of the pocket gophers of the subfamily Geomyinae. Univ. Kans. Pub., Mus. Nat. Hist. 16: 473-579. Saiki, R. K., Bugawan, T. L., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1986). Analysis of enzymatically amplified 8-globin and HLA-DQa DNA with allele-specific oligonucleotide probes. Nature 324: 163-166. Saiki, R. K., Gelfand, D. H., Stoffel, S. , Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primerdirected enzymatic amplification of DNA with a thermostable DNA Polymerase. Science 239: 487-491. Sanger, F., Nicklon, S., and Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467.
Shields, G. F., and Wilson, A. C. (19871. Calibration of mitochondrial DNA evolution in geese. J. Mol. Evol. 24: 212-217. Smith, M. F., and Patton, J. L. (1991). Variation in mitochondrial cytochrome b sequence in natural populations of South American Akodontine rodents (Muridae: Sigmodontinae). Mol. Biol. Evol. 8: 85-103.
Swofford, D. L. (1989). “PAUP: Phylogenetic Analysis Using Parsimony,” Illinois Natural History Survey, Champaign, IL.
PHYLOGENETICS
AND
EVOLUTION
Vol. 1, No. 1, March, pp. 17-25, 1992
Phylogenetic Relationships among Middle American Pocket Gophers (Genus Orthogeomys) Based on Mitochondrial DNA Sequences PHILIP
D. SUDMAN AND MARK 5. HAFNER
Museum of Natural Science and Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803-32 76 Received August 23, 1991; revised February 3, 1992
interspecific interactions, phylogenetic relationships, or even the basic biology of these rodents. The fossil record of Orthogeomys is meager, with only a single late Pleistocene specimen known [O. onerosus (Russell, 1960, 1968)l. In the absence of fossil data from which to reconstruct the phylogenetic history of the Orthogeomys lineage, relationships among the extant taxa have been inferred from genetic data. Hafner (1982) demonstrated that the three subgenera are well differentiated from each other based on both protein-electrophoretic and immunological evidence. However, the relationships among these taxa remain unclear. Phenetic analyses of electrophoretic data suggest that the subgenera Orthogeomys and Heterogeomys are sister taxa, whereas cladistic (locus-by-locus) analyses and immunological data indicate that Mucrogeomys is more closely related to Heterogeomys (Hafner, 1979, 1982). Relationships among Orthogeomys at the species level also are unresolved, especially within the subgenus Macrogeomys (Hafner, 1991); each of the other subgenera contains only two species. Protein-electrophoretic data are strongly indicative of a sister&axon relationship between 0. cherriei and 0. heterodus and between 0. cauator and 0. duriensis. However, the placement of 0. underwoodi within Macrogeomys could not be resolved definitively (Hafner, 1991). Cladistic analysis placed 0. underwoodi as a sister taxon to the cherriei-heterodus lineage, whereas parsimony analyses of the same data based on the presence-absence of alleles ally 0. underwoodi with the cavator-dariensis clade. The sixth species within this subgenus, 0. matugulpae, was not available for analysis, and the placement of a newly described member of the subgenus, 0. thueleri (Alberico, 1990), has yet to be determined. In an effort to better understand the relationships within and among the subgenera of Orthogeomys, we used nucleic acid sequence data from two regions of the mitochondrial genome for phylogenetic analyses. These results are compared with those of similar stud-
Relationships among members representing each of the three subgenera of the Middle American rodent genus Orthogeomys (Rodentia: Geomyidae) were studied by comparing DNA sequence data from two regions of the mitochondrial genome. Results from 527 bp from the 16 S rDNA region and a 402.bp fragment of the cytochrome b gene indicate that the three subgenera are well differentiated genetically, with the subgenus Orthogeomys being distantly related to Macrogeomys and Heterogeomys, and Macrogeomys appearing as the most derived. Within the subgenus Macrogeomys, 0. he&o&s and 0. cherriei form a distinct clade, as do 0. dariensis and 0. cavator. As with previous proteinelectrophoretic studies, the placement of 0. underwood could not be determined definitively within the subgenus Macrogeomys. We interpret our inability to determine phylogenetic relationships among these three clades as evidence for a rapid phyletic radiation within this subgenus. Sequence divergence estimates indicate that the Macrogeomys radiation took place following the time of completion of the Panamanian land bridge (1.9-2.9 myal. Additionally, the near identity of sequences of a newly described species, 0. thaeleri, with those of 0. dariensis (percentage sequence divergence = 0.3%) suggests that the two may be conspecific. B 1X42 Academic Prees, Inc.
INTRODUCTION
Middle
American
pocket gophers of the genus Ormembers of the rodent family Geomyidae. The genus, which ranges from Mexico south to northwestern Colombia and occupies a wide range of habitats, comprises three subgenera, Orthogeomys, Heterogeomys, and Macrogeomys. Because of their subterranean habits and the relative inaccessibility of major portions of their geographic ranges, very little is known about the distributions, thogeomys are among the least-studied
17 1055-7903/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
18
SUDMAN
AND
ies based on protein electrophoresis to elucidate degrees of divergence, patterns of descent, congruence of evolution in different (independent) genomic regions, and specific status of the taxa in question. MATERIALS
AND
METHODS
Total nucleic acid preparations were obtained from liver or kidney following the phenol/chloroform extraction techniques summarized by Hillis et al. (1990). Amplifications of specific regions of the mitochondrial DNA (mtDNA) genome were then performed via the polymerase chain reaction (PCR) using the following sets of primers: 16 S rDNA, L2510 (5’-GGAATTCCCGCCTGTTTATCAAAAACAT-3’) and H3062 (5’~GGAATTCCCTCCGGTTTGAACTCAGATC-3’); cytochrome b, L14724 (5’-CGAAGCTTGATATGAAAAACCATCGTTG-3’) and H15149 (5’-CCTCAGAATGATATTTGTCCTCA-3’). L and H refer to the 3’ position of the primers in relation to human mtDNA light and heavy strands, respectively (Anderson et al. 1981). Double-stranded PCR amplifications using Thermus acquaticus DNA polymerase (Saiki et al., 1986, 1988) were performed in 50 or loo-k1 total reaction volumes following the balanced primer procedure described by Allard et al. (1991). Twenty-five to 35 cycles were performed with the following cycling parameters: 16 S rDNA-1 min denaturation at 93°C 1 min annealing at 48”C, and 1 min extension at 72°C; cytochrome b-l min denaturation at 93”C, 1 min annealing at 56”C, and 1 min extension at 72°C. Five microliters of the double-stranded product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized under UV to assess reaction success. Five microliters of the double-stranded product was then used to generate single-stranded DNA in loo-k1 reactions following the same procedures outlined above, except that only one primer was added to the reaction mixture. Single-stranded DNA was generated for both the heavy and the light mtDNA strands, and the products were cleaned by multiple washings with H,O through Ultrafree-MC 30,000 NMWL filters (Millipore Corp., Bedford, MA) and concentrated to a final volume of 50 p-1. Seven microliters of the cleaned single-stranded templates was then used for DNA sequencing using T7 DNA polymerase (Sequenase version 2.0, United States Biochemical, Cleveland, OH) following standard dideoxy chain termination protocols (Sanger, 1977). Both mtDNA strands were sequenced to within approximately 30 bp of the sequencing primer. Sequences were aligned using the ALIGN software program (version 1.0, Scientific & Educational Software, 1989) and trees were constructed using PAUP, version 3.0 (Swofford, 1989) and PHYLIP, version 3.3 (Felsenstein, 1990). Because of uncertainty about the
HAFNER
relationships among genera within the tribe Geomyini (Hafner, 1982; Honeycutt and Williams, 1982), Thomomys talpoides (tribe Thomomyini) was used as an outgroup for all analyses. Tree-building procedures included maximum parsimony (Fitch, 1971), maximumlikelihood (Felsenstein, 1981), and Fitch and Margoliash (1967) methods for fitting trees to distance matrices generated following the assumptions of the Kimura two-parameter model (Kimura, 1980). Analyses were performed for the entire data set as well as for transversions only. Additionally, Mantel’s test (Mantel, 1967) was used to compare a matrix of genetic distance values from protein data (Hafner, 1991) with the percentage sequence divergence estimates generated in this study to determine the relationship between nuclear and mitochondrial data sets. The following specimens were examined: Orthogeomys hispidus-Louisiana State University Museum of Natural Science (LSUMZ) 29232 0 ; Belize: Cayo District, 3 km W Belmopan. Orthogeomys thaeleri-LSUMZ 30057 0 ; Colombia: Departamento de1 Chock, Ensenada de Utria, Parque National Natural Utria, Municipio de Bahia Solano, -10 m. Orthogeomys cavator-LSUMZ 29490 8 ; Costa Rica: Prov. San Jose, 3 km SW Division, 2200 m. Orthogeomys cherriei-LSUMZ 29539 6 ; Costa Rica: Prov. Guanacaste, 2 km E Tronadora. Orthogeomys heterodus-LSUMZ 29498 0 ; Costa Rica: Prov. Cartago, 2 km W Santa Rosa, 2300 m. Orthogeomys underwoodi-LSUMZ 29537 9 ; Costa Rica: Prov. Alajuela, 3 km S Orotina. Orthogeomys grandis-Museum of Vertebrate Zoology (MVZ) 154079 c?; Mexico: Michoacan, 3.6 km (by road) N Arteaga. Orthogeomys dariensis-LSUMZ 25436 d ; Panama: Prov. Darien, 6 km SW Cana, 1200 m. Thomomys talpoides-LSUMZ 29301 Q; New Mexico: Sandoval Co., 3 km N, 17.8 km E Jemez Springs. RESULTS
Alignment of the sequences resulted in a final data set of 929 bp (Fig. l), including 527 bp from 16 S rDNA [homologous to the region between human positions 2550 and 3062 (Anderson et al., 198l)l and 402 bp from the cytochrome b gene coding region [human positions 14,747 to 15,149 (Anderson et al., 1981)]. The sequences from 0. duriensis and 0. thaeleri were identical, with the exception of three transitions, one within the cytochrome b sequence and two within the 16 S rDNA sequence (see asterisks, Fig. 1); 0. thaeleri was therefore omitted from all further analyses. The majority of variation within the 16 S rDNA was partitioned within presumed hypervariable regions, whereas variation within the portion of the cytochrome
PHYLOGENETIC
RELATIONSHIPS
AMONG
19
Orthogeomys
>>16S rDNA AGTGACAAAA GTTAAACGGC CGCGGTATTC TGACCGTGCA AAGGTAGCAT AATCATTTGT . .. .. . . .......... .......... .......... .......... .......... .. ... . . .......... .......... .......... .......... .......... . .. .. . . .......... .......... .......... .......... .......... .......... . .. .. . . .......... .......... .......... .......... . . ... . . .......... .......... .......... .......... .......... .. ... . . .......... .......... .......... .......... .......... .. ... . . .......... .......... .......... .......... .......... .. ... . . .......... ........ c. .......... .......... .T...C ....
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
0. 0. 0. 0, 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
TCTCTAATTA AGGACTTGTA TGAATGGCAC GACGAGGGTT .......... .. A ........... C ............... .......... ...... C ........... G. A....A .... .......... .............................. .......... .............................. .......... .............. C ............... .......... .............. C ............... .......... .............. C ............... . . . T . . . . . . .................. GT ..........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
TCTATGAAAT TGACCTTTCC GTGAAGAGGC GGAAATAAAT AAATAAGACG .......... .......... .......... .......... .......... . . A ....... .... T ..... .......... .......... T ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TTCA T .........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
ATGGAGCTTC .......... . . . . . . . . .T .......... .......... .......... .......... .......... . . . . . . . . .A
AATTAAA-T.......... .G....T... .......... .......... .......... . . . . .T . . . . . . . . .T . . . . . . . . . . TA.A
ACTTTATAAT . . . . . . . . .C . ..C....C. . . . . . . . . .C . . . . . . . . .C . . . . . . . . .C . . . . . . . . .C . . . . . . . ..c G..C.TCTTA
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
AATAACTAAC ......... T ...... ..-. ......... T ......... T ......... T ......... T ......... T ... ..- ... A
TTAA-AAGT .......... ...... G ... .......... .......... .. T ....... .......... .......... -G..AAG ...
AAAAAA-TTT TGGTTGGGGT GACCTCGGAG CACAACAAAA ........................................ T ... ..AC ...................... ..T.G ..... ........................................ ........................................ .................................. G ..... ....................... . ........ ..G.G ... ................................ ..G..C .. T .. ..- ........................ T.T.GT . ..C
TTACTGTCTC TTACATTCAA ............. T ... T .. .A .................. ............. T ... T .. ............. T ... T .. ............. T ... T .. ............. T ... T .. ............. T ... T .. .A ........ .C.T...TT. AGAAGACCCT ...... .. ...... .. ...... .. ...... .. ...... .. ...... .. ...... .. ...... ..
* TTTAAATATT . . . . . ..TCA C.....A.CA . . . . . . . . CA . . . . . . . . CA C.......CA . . . . . . c.AA c.....c.AA .GA.TT..AA
ACTCCA-CAG . . ..T....A ..CTA..... . . ..T....A . . ..T.... G .......... . . . . . . ...* .......... .A..A.AT.A
GAA-ATAACA .......... . ..T.C.... .......... .......... . . ..G..... I......... .......... ..G.......
FIG. 1. Aligned sequences for each of the nine species of pocket gophers (Orthogeomys and Thomornys) analyzed. Dots indicate identity with the 0. h&i&s sequence as depicted in the first row, and dashes indicate gaps inserted to maximize homologies. Asterisks designate the three positions where 0. dariensis and 0. thaeleri differ in sequence.
b gene was evenly dispersed throughout. Within Orthogeomys, 65 of the 527 16 S rDNA positions were variable; 67.2% of these differences were transitions, and 32.8% were transversions. Within the cytochrome b region, 105 of 402 sites were variable within Orthogeomys (73.3% transitions and 26.7% transversions), resulting in amino acid changes at 13 of the 134 codons. The distribution of the variable cytochrome b sites with respect to first,
second, and third codon positions was 20.0, 4.8, and 75.2%, respectively. Percentages of silent and replacement transitions and transversions for each position are given in Table 1. The 0. underwoodi sequence was unique in possessing a 3-bp deletion at the third or fourth amino acid position. Although deletions have not been previously reported in this region of the cytochrome b genome (Irwin et al., 1991; Howell, 1989; Smith and Patton, 19911, codon deletions and additions
20
SUDMAN
AND
HAFNER
* 0. 0. 0. 0. 0. 0, 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
CCTCCGAAAG ACTATTATT- TAGACCTGCA AGTCAACATT AG..A. C ...... A ............ .............. .............. A . ..A T ....... A ............ .......... .T..AA..A. ....... A ...... G.T ... .......... .T..AA..A. C ...... A ...... G.T ... A ............ ............ ..AG..C. ....... .............. AG .... .. ..A.AA ............ .............. AG .... .... A.AA ............ .............. AC .... ...... CA ...... ..T ...
-AACATAA-G ... T ..... A ... T ...... ... T.C .. .A . ..T.C . ..A ... T ..... A ... T ..... A ... T ..... A TT.T-...A.
T-CCT-AATT ... AC ..... C..T ...... .T.A ...... .T.A ...... ... AC ..... ... AC ..... ... AC ..... . ..T.A ....
TAACAGCGCA ATCTTATTGG
0.
cavator
0. 0. 0. 0, 0. 0. T.
grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
GACCCAGAAA TCTGATCAAT GGACCAAGTT ACCCTAGGGA .................... .A .................. .................... .................... .................... .A .................. .................... .A .................. .................... .A .................. .................... .A .................. .................... .A .................. .. T ....... C ......... ....................
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
AGAGTCCATA TCGATAATAA GGTTTACGTA CTCGATGTTG GATCAGGACA TCCTAATGGT ............................ AC .................... ...... C ... ............................ AC .................... .......... ............................ AC .................... .......... .......................... ..A C .................... .......... ............................ AC .................... .......... ................... G ...... ..A C .................... .......... .......................... ..A T .................... .......... .......... ..... T ................ C ..... AC ....................
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
GCAGAAGCTA .......... .......... .......... .......... .......... .......... .......... ..........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
CTTTTAGAGT GGGTGATTAT ....... G .. A..C..A ... ....... T ..... AA ..... ....... G .. A..C..A ... ....... G .. A..C..A ... ....... G ........ C ... ....... G .. A. ........ ....... G .. A ......... .A...T.T ... ..GA..A .A
0. hispidus
TTAAGGGTTC .......... .... T ..... .......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... .......... .......... ..........
.......... .......... .......... .......... .......... .......... .......... ..........
16s I-DNA cytochrome h GTTTGTTCAA CGATTAAAGT CCTACGTTAC TGTTAATATG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..T.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GG.. . ..T
................................. ...................................
.... T ....
T---. ................................... ............................ G. ... ..C .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. . . . . . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . .G. . . .
TTTTAACAGT .......... .......... .......... .......... .......... ........ A. .......... ..........
TGGTACGAM GTAACTGGAT GGTTGTGGTG A .......... ....... G .. ......... .......... ... G .............. G. .......... ... G ................ .......... ... G ................ .......... ... G .............. G. ....... G .. ... G ... A ............ .......... .C.G ................ .. ..G..T .. A ......... ..A..G ....
FIG. l-Continued
are known to occur within the cytochrome b coding region in other mammals (Irwin et al., 1991). Uncertainty about the exact position of the deletion exists because both the third and the fourth amino acid codons are identical in three of the Orthogeomys species examined. That this deletion is widespread was confirmed by examining the sequences of three additional specimens of 0. underwoodi from throughout its range. All three specimens contained identical sequences within the region in question. Regardless of which tree-building algorithm was
used, the cytochrome b data set yielded a single tree with the same topology as that shown in Fig. 2a. Independent analysis of the 16 S rDNA data yielded three trees, all, of which were identical to the cytochrome b tree except for the replacement of 0. underwoodi within the subgenus Mucrogeomys. Because of this overall similarity, the cytochrome b and 16 S rDNA data sets were combined for all remaining analyses. Percentage sequence divergence within Orthogeomys ranged from 2.77 to 13.56, with 0. grandis showing the greatest degree of divergence from all other
PHYLOGENETIC
RELATIONSHIPS
TCCAACTACT ..C ... ..C GT ........... G..T ...... G..T ...... T ...... ..T ...... ..T ...... ..T.A .......
AMONG
TTAAAGCCGA ..G..A .... ..A..A. ..G..A .... ..G..A .... ..G..A .... ..G..A..A. ..G..A..A. ..A..C.
21
Orthogeomys
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
GATTATAGAG .T..G ...... .... G ..... .G..G..A .. .G..G..A .. .... G ........ .G..G ...... .G..G ...... .... G ......
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus oherriei talpoides
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
AGAGATGTCC GGATAAAGAT CGTTATGTGA TGTGTAGACT GTGTGAATGT CGGAAGAGTA .A..T ........... ..A ............ .A.....T ............ ..A.....C. . ..AT ..... .A....GA ................. ..T ... ..G..G ... ..A ....... .... T ............ A ............ .A..C..T ............ ..A.....C. .... T ............ A ............ .A..C..T ............ ..A.....C. .A..T .......... ..A .. ..A ............ ..T .. A ...... ..A ..A ....... .A..T .......... ..A .............. ..C..T ............ ..A ....... .A..T .......... ..A .............. ..A ....... ..C..T ............ ... AT..G .. T.....G ... ..A.....A. ..... ..T ... ..G ...... ..A..A .... * GACATCGTGT GTAAACGGCA CTGCATTTGA TGCCGACTAA TTAGGCTATG TATGTACGGT .T ................. . ............ ..T .............. . .......... .T...T .... A ...... ..T ..A ......... ..T ...... ..A ................ .T...T ............. . ......... . .. ..T .............. . G ......... .T...T .... A ...... ..G ......... . .. ..T .............. . G ......... .T ........ A ...... ..G ......... . .............................. . .......... .T ................. . ............................. . .......... .T ........................... . A .................. .T..AT.A .. A ...... ..T ..A ....... ..A ... ..A ...... ..A. .A .........
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
TGCCGCGGAG GGATAAGAAG .A..A ..... A ......... .A.....A ............ .A..A ............... .A..A ............... .A..A ..... .A ........ .... A ............... .A..A ............... .... T ..... A ........ A
TAAACGGAAA .......... . . G . . . . . G. .......... .......... .......... . . . . . . . . G. .*... A . . . . . . . . ..A.T.
TATAAGTGTA .G........ .G..G..A.. .G........ .G........ .G........ .G........ .G........ .G..G..A..
GCCTGCTCCT .................. ......... A .................. .................. ......... C ......... G ......... G A ..............
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thael eri underwoodi heterodus cherriei talpoides
CTAGGATAGA . . . .A . . . . . . . . . A . . . A. . . . .A. . . . . . . . .A . . . . . . . . .A . . . . . . . . . . 0. G . . .C.....G.. . . . . . . . G. .
AATATGTCTT .......... T..GA..... .......... . ..*...... G..G.T.... G . . . . T.... . . . . .T . . . . . . . . .T . . . .
TGTACTTTGT ........ A. .......... ... ..C..A. ... ..C..A. ........ A. ........ A. ........ A. ........ A.
AGCCGTAAAA ........... .A..T..GG. .T ........ .T ........ .......... ........... .A ............ .A.....G ..
TAATGATAAA AATTGATATC ..CA .. ..G .......... CG..A .. ..G G .......... .G.C ... ..G .......... .G.C ... ..G .......... .G ..... ..G G....G .... ..C ................ ..C ............. .G ........... ..T ....
0. 0. 0. 0. 0. 0. 0. 0. T.
hispidus cavator grandis dariensis thaeleri underwoodi heterodus cherriei talpoides
GGTGACGTAA .......... .T........ .......... . ...*..... . . . . . . .C. . .......... .......... .A..T..G..
GCATCCTATA CT........ A..C..A..G .T........ .T........ .T........ .T........ .T.......G
CATGATGGT . . . ..C... ......... ..c..c... ..C..C... ......... . . . ..C... . . . . .C. . .
A.....A...
..GA.....
FIG. l-Continued
GGGATAATCC . . . . . G. . . . .......... . . . . . G. . . . . . . . . G. . . . . . . . . G. . . . . . ..CG.... . . . . . G. . . . . . ..CG.A..
TGATACGAAT .......... .... ..AG .. .......... .......... .......... .......... .......... .T ........
TAAGATGTTT ... A ...... .GG..A .... . .GA ...... ..GA ...... ... A ...... ... A ...... ... A ...... ..........
TAAATGATAC G. .. G ....... G. G. .......... .......... .......... A ....
22
SUDMAN
TABLE
AND HAFNER
1
Distribution of Silent and Replacement Nucleotide Changes for Pairwise Comparisons between Seven Species of Orthogeomys at 402 Sites within the Cytochrome b Gene Codon position
Transitions Silent Replacement Transversions Silent Replacement
First
Second
Third
95 (10.6) 49 (5.5)
0 (0.0) 18 (2.0)
499 65.8) 10 (1.1)
5 (0.6)
0 (0.0)
12 (1.3)
18 (2.0)
8.)
(19.1) 18 (2.0)
170
Note. Percentages are given in parentheses.
taxa (Table 2). Based on sequence divergence alone, the subgenus Orthogeomys (represented by 0. grandis) is the most divergent. The subgenera Macrogeomys and Heterogeomys (represented by 0. hispidus) display a moderate degree of similarity (average percentage sequence divergence = 7.26). Within the subgenus Macrogeomys, sequence divergence ranges from 2.77 between 0. cherriei and 0. heterodus to 5.92 between 0. cherriei and 0. dariensis. Results of the Mantel’s test comparing the percentage sequence divergence values (this study) with Nei’s (1978) unbiased genetic distance (Table 2) computed from the protein electrophoretic data of Hafner (1991), indicated a high level of concordance between the two matrices (t = 2.15, zvO.05). In general, transition-to-transversion ratios (Table 3) decreased with increasing sequence divergence. Within the subgenus Macrogeomys, ratios ranged from 2.00 to 5.25, whereas ratios among the three subgenera of Orthogeomys ranged from 2.00 to 2.88. Transitionto-transversion ratios were lowest for comparisons between Orthogeomys and Thomomys (ranging from 1.03 to 1.28), supporting the general assumption that transitions saturate over time (Brown, 1982). All three tree-building procedures resulted in the same interpretation of the relationships among the three subgenera of Orthogeomys, regardless of whether they were based on the entire data set or transversions only. Based on these analyses, the subgenera Heterogeomys and Macrogeomys are sister taxa within the genus Orthogeomys, and show a closer association to each other than either does to the subgenus Orthogeomys. Members of the subgenus Macrogeomys show a high degree of overall similarity. The position of 0. underwoodi within the subgenus Macrogeomys differed depending on the method of analysis. In all of the analyses, 0. cherriei and 0. h&rodus were depicted as sister taxa, as were 0. dariensis and 0. cavator. 0. underwoodi was portrayed as the
I
13 ati 44
T.ta#waea
b.)
FIG. 2. Phylograms resulting from parsimony analyses of mtDNA data depicting relationships within the genus Orthogeomys. Thomomys tulpoides was included as an outgroup. The numbers on each branch indicate branch lengths. (a) The most-parsimonious tree based on analyses of all mutations. The consistency index (CI) for the tree is 0.825. Although this topology was supported by maximum-likelihood analysis, a Fitch-Margoliash analysis of distance values placed 0. underwoodi as a sister taxon to the 0. chrriei-0. heterodus clade as in Fig. lb. (b) Most-parsimonious tree when only transverions were used in the analyses (CI = 0.896). The topology of this tree was supported by all three tree-building algorithms.
outgroup of all other Macrogeomys in both maximum likelihood and parsimony analyses (Fig. 2a), but was placed as a sister group to the cherriei-heterodus clade in the Fitch-Margoliash analysis of distance values. It should be noted, however, that a parsimony tree containing the same topology as that obtained from the distance analysis (i.e., 0. underwoodi allied with the cherriei-heterodus clade) was only three steps longer than the most-parsimonious tree (CI = 0.817), and one depicting 0. underwoodi as the sister taxon to the cauator-dariensis clade was only two steps longer (CI = 0.820). Trees based on transversion data alone were more consistent than those derived from both transitions and transversions (Fig. 2b). All three tree-building algorithms were in agreement in the placement of 0. underwoodi as the sister group to 0. cherriei and 0. heterodus. However, a parsimony tree one step longer than that illustrated in Fig. 2b depicted the relation-
PHYLOGENETIC
RELATIONSHIPS
TABLE
AMONG
23
Orthogeomys
2
Percentage Sequence Divergence (Rimura, 1980) for Orthogeomys Taxa and the Outgroup, Thomomys, Derived from Comparison of 929 bp from Two mtDNA Regions (above Diagonal) and Nei’s Unbiased Genetic Distance (Nei, 1978) for the Same Taxa Computed from the Data of Hafner (1991) (below Diagonal) 1 1 2 3 4 5 6 7 8
0. 0. 0. 0. 0. 0. 0. T.
gmndis hispidus cavator cherriei dariensis heterodus underwoodi tulpoides
Note.
Thomomys
0.693 1.135 0.921 1.217 0.937 0.992 talpoides
was not
2
3
4
11.39 1.067 0.804 0.958 0.947 0.837 -
13.17 7.23 0.195 0.256 0.229 0.193 -
13.48 7.25 5.81 0.305 0.093 0.092 -
included
in the study
by Hafner
ships within the subgenus Mucrogeomys as an unresolved trichotomy between 0. underwoodi and the cavator-dariensis and cherriei-heterodus clades (CI = 0.888).
DISCUSSION In his revision of the subfamily Geomyinae, Russell (1968) synonymized the genera Orthogeomys, Heterogeomys, and Macrogeomys into the genus Orthogeomys. Although he retained each of the former genera as subgenera, Russell stated: “A revision of the genus is needed; it might show that the currently recognized subgenera are artificial, and that a different arrangement of the species would more clearly express their evolutionary relationships” (Russell, 1968, p. 510). Based on the sequence data presented herein and the protein data of Hafner (1982), each of the subgenera appears to be genetically well differentiated, at least within the context of the species examined. Although the relationships among the subgenera were not readily apparent from previous work based on protein data, mtDNA sequence data indicate that Mucrogeomys and Heterogeomys are more closely related to each other than either is to Orthogeomys. TABLE
1 2 3 4 5 6 7 8
0. 0. 0. 0. 0. 0. 0. T.
gmndis hispidus cavator cherriei dariensis heterodus underwoodi talpoides
Note.
Transitions/trausversions
Pairwise
Comparisons 929.bp
1
2
2.00 2.44 2.03 2.62 2.42 2.00 1.28
64132 2.50 2.00 2.88 2.59 2.50 1.03 are
shown
78132 45118 2.00 3.57 2.38 2.36 1.08 the
diagonal
13.56 6.99 4.97 2.77 5.67 0.143 -
7
8
11.82 7.26 5.34 4.75 5.69 5.10 -
16.30 15.95 17.21 16.92 16.92 16.05 16.81 -
(1991).
Interspecific relationships within the subgenus Mczcas determined by protein electrophoresis (Hafner, 1991) are well supported by all analyses of the mtDNA data. 0. cherriei and 0. heterodus are characterized as being the least differentiated from each other in terms of their percentage sequence divergence and absolute numbers of transitions and transversions. 0. cavator and 0. dariensis also display close affinities at all levels, with slightly higher percentage sequence divergence and numbers of transitions and transversions than the cherriei-heterodus clade. As with analyses of protein data, the placement of 0. underwoodi within the subgenus Mucrogeomys could not be determined definitively. The phylogenetic placement of 0. underwoodi within Macrogeomys therefore remains unresolved, and our present understanding of the relationships within the subgenus can best be represented as an unresolved trichotomy involving 0. underwoodi and the cavator-dariensis and cherriei-heterodus lineages. It could be argued that our inability to resolve completely evolutionary relationships within the subgenus Mucrogeomys is simply the result of an insufficient number of informative sites in the mtDNA data set. It seems more likely, however, that the lack of resolution rogeomys
3 Based
on the
4
5
6
75137 42121 34117
76129 49117 2517 36/16
80133 44117 31113 2114 38112 3.09 1.11
2.25 5.25 2.82 1.08 and
6
12.50 7.58 3.57 5.92 0.378 0.272 -
of Transitions and Transversions mtDNA Sequences Reported Herein 3
above
5
transition-to-transversion
3.17 2.85 1.08 ratios
below
7 66133 45118 33114 31111 37113 34111 1.16 the
diagonal.
8 74158 66164 71166 70165 70165 68/61 72162 -
24
SUDMAN
is actually evidence for a rapid phyletic radiation within the subgenus. Kraus and Miyamoto (1991) suggest that short internode lengths and near identical levels of sequence divergence among taxa are consistent with hypotheses of rapid origin and radiation of lineages. As can be seen in the parsimony trees (Figs. 2a and 2b), the internode lengths between the three lineages of Macrogeomys are relatively short compared to the terminal branches. The other two tree-building algorithms used in this study support these observations; in each case, internodal lengths between the lineages of Macrogeomys are small compared to terminal branch lengths. Estimates of sequence divergence between 0. underwoodi and the other species of Macrogeomys (Table 2) are similar, as are the absolute numbers of transversions (Table 3). Together this evidence seems to point toward a rapid radiation involving the ancestors of each of the cauator-dariensis, cherriei-heterodus, and 0. underwoodi lineages. In an effort to resolve more clearly the relationships within Macrogeomys, we used a jackknifing procedure (Lanyon, 1985) with each of the tree-building algorithms. Lanyon (1985) advocated use of a jackknife approach as a means of identifying internal inconsistencies within a data set. In each case, jackknifing indicated that relationships within the subgenus Macrogeomys were best represented as an unresolved trichotomy between 0. underwoodi and the cauatordariensis and cherriei-heterodus clades. Studies based on protein-electrophoretic data also failed to resolve completely the relationships between 0. underwoodi and the other members of the subgenus (Hafner, 1991). Thus, studies of the nuclear genome (Hafner, 1991) are concordant with studies of the mitochondrial genome (this study) and, together, they suggest a rapid phyletic radiation early in the history of the subgenus Mucrogeomys, because both nuclear and mitochondrial DNA data sets failed to resolve unequivocally relationships within the subgenus. Brown et al. (1979) and Shields and Wilson (1987) have suggested that vertebrate mtDNA evolves at a rate of approximately 2% per million years. Using this as a rough estimate of divergence rates, we calculated times since divergence based on our data (Table 2). Estimates of time since divergence between Orthogeomys and Thomomys places the cladogenic event between the tribes Geomyini and Thomomyini at approximately 8 million years ago (mya). This is in close agreement with divergence times estimated from the fossil record, which indicates that Thomomys split from the main geomyid lineage during the late Miocene (Russell, 1968). Within the genus Orthogeomys, sequence data indicate that the subgenus Orthogeomys diverged from the Macrogeomys-Heterogeomys lineage approximately 6 mya, and that Macrogeomys and Heterogeomys diverged from each other approximately 3.6 mya. The
AND
HAFNER
first of these estimates is in close agreement with Russell’s (1968) proposed timing of the major radiation within the tribe Geomyini during the mid Pliocene. Our estimate of time since divergence of Heterogeomys and Macrogeomys (3.6 mya) places this event contemporaneous with the completion of the Panamanian land bridge (Marshall et al., 1979). Considering that the present distribution of Mucrogeomys straddles the Panamanian isthmus, it seems reasonable that the principal phyletic radiation of this group was triggered by major geologic events in the region, including the availability of new habitat resulting from geologic uplift. Divergence estimates within the subgenus Macrogeomys are on the order of 1.9 to 2.9 mya. Although these time estimates are considerably more recent than those estimated by Hafner (1991) based on Nei’s genetic distances (4.5 mya), they are well within the proposed historical biogeographic framework proposed for the subgenus (Hafner, 1991). Hafner (1991) speculated that the current distributions and relationships within the subgenus may be directly related to isolating effects of repeated glacial and interglacial periods of the Quaternary. These events would have essentially transformed the mountainous regions of Middle America into a series of islands, resulting in repeated fragmentation, and perhaps speciation, of ancestral populations. Divergence estimates roughly corresponding to the early Pleistocene (this study) are consistent with this hypothesis. 0. thaeleri, originally described as distinct from 0. dariensis based on pelage and cranial differences (Alberico, 19901, occupies a small range in the northwestern corner of Colombia within 50 km of the known distribution of 0. dariensis. Pocket gophers have been shown to exhibit a high degree of morphological variation over short geographic distances due to local habitat differences (Patton and Brylski, 19871. It is possible, therefore, that populations representative of 0. dariensis and 0. thaeleri are distinct morphologically, but are, nevertheless, members of a single gene pool. 0. thaeleri and 0. dariensis had an estimated sequence divergence of only 0.3%, whereas all other intrageneric comparisons exceeded 2.7%. Irwin et al. (1991) reported intraspecific sequence variation of 0 and 1.7% for cow @OS tuurus) and long-beaked dolphin (SteneZZa Zongirostris), respectively, over the entire cytochrome b gene. Smith and Patton (1991) reported that sequence differences among conspecific populations of the rodent genus.Akodon ranged from 0.25 to 8% based on the same portion of the cytochrome b gene examined in this study. Although it is certainly not valid to make taxonomic decisions based solely on percentage sequence divergence, we feel that because the estimate of mtDNA sequence divergence between 0. thaeleri and 0. dariensis falls well within the range of values reported for conspecific populations of other mammals,
PHYLOGENETIC
RELATIONSHIPS
Q. thaeleri may represent a geographic variant of 0. duriensis. However, additional sampling from the region between the two forms is needed before formal taxonomic changes can be considered. ACKNOWLEDGMENTS We thank J. Spatafora for technical assistance with PCR and sequencing protocols, J. Derr and S. Davis for the 16 S rDNA primers, M. Alberico for tissue samples from 0. thueleri, D. J. Hafner for specimen collection, and J. Demastes, D. Good, and R. Zink for helpful comments and criticisms of earlier drafts of the manuscript. This research was supported by National Science Foundation Grant BSR-8817329.
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