Exp Appl Acarol DOI 10.1007/s10493-015-9939-7

Post-embryonic development in the mite suborder Opilioacarida, with notes on segmental homology in Parasitiformes (Arachnida) Hans Klompen1 • Ma. Magdalena Va´zquez2 Leopoldo Ferreira de Oliveira Bernardi3

•

Received: 15 March 2015 / Accepted: 11 June 2015 Ó Springer International Publishing Switzerland 2015

Abstract In order to study homology among the major lineages of the mite (super)order Parasitiformes, developmental patterns in Opilioacarida are documented, emphasizing morphology of the earliest, post-embryonic instars. Developmental patterns are summarized for all external body structures, based on examination of material in four different genera. Development includes an egg, a 6-legged prelarva and larva, three 8-legged nymphal instars, and the adults, for the most complete ontogenetic sequence in Parasitiformes. The prelarva and larva appear to be non-feeding. Examination of cuticular structures over ontogeny allows development of an updated model for body segmentation and sensillar distribution patterns in Opilioacarida. This model includes a body made up of a well-developed ocular segment plus at most 17 additional segments. In the larvae and protonymphs each segment may carry up to six pairs of sensilla (setae or lyrifissures) arranged is distinct series (J, Z, S, Sv, Zv, Jv). The postprotonymphal instars add two more series (R and Rv) but no extra segments. This basic model is compatible with sensillar patterns in other Parasitiformes, leading to the hypothesis that all taxa in that (super)order may have the same segmental ground plan. The substantial segmental distortion implied in the model can be explained using a single process involving differential growth in the coxal regions of all appendage-bearing segments.

& Hans Klompen [email protected] Ma. Magdalena Va´zquez [email protected] Leopoldo Ferreira de Oliveira Bernardi [email protected] 1

Acarology Laboratory, Ohio State University, 1315 Kinnear Road, Columbus, OH 43212-1192, USA

2

Division de Ciencias e Ingenierias, Universidad de Quintana Roo, Chetumal, Quintana Roo, Mexico

3

Departamento de Biologia, UFLA, Lavras, Minas Gerais, Brazil

123

Exp Appl Acarol

Keywords Segmentation
Opilioacarida
Ixodidae
Mesostigmata
Ontogeny
Differential growth

Introduction The order (Lindquist et al. 2009) or suborder (Norton et al. 1993) Opilioacarida has always occupied a special place in studies of mite evolution, out of proportion to its relatively small taxonomic diversity. In general appearance, the soft-bodied Opilioacarida resemble small cyphophthalmid Opiliones or large eupodoid Acariformes (the larger mite lineage) more than they do other Parasitiformes (Ixodida, Holothyrida, Mesostigmata). They share the presence of an ovipositor, eugenital setae, a prelarva, a distinct rutellum, and solid feeding mode with many Acariformes [nearly all primitive characteristics or of uncertain homology (Dunlop and Alberti 2007; Lindquist 1984)], and the presence of a basitarsus on legs II–IV, a distinct cluster of subdistal sensilla on tarsus I (elaborated into Haller’s organ in Ixodida), and the structure of the spermatozoa (Alberti 1980, 2005) with Parasitiformes. On occasion Opilioacarida have been assigned to a rank (Opilioacariformes) equivalent to that of Parasitiformes and Acariformes (e.g. Grandjean 1969; Johnston 1982; Walter and Proctor 1999), but recent molecular studies clearly support the hypothesis that the group is part of Parasitiformes (Klompen 2010; Klompen et al. 2007; Murrell et al. 2005; Pepato et al. 2010). In order to improve understanding of Opilioacarida, and to possibly find additional morphological or developmental characters shared with other Parasitiformes, the current study is aimed at documenting developmental patterns in the group. Broad aspects of development in Opilioacarida are already well known: Coineau (1973) established the presence of a prelarva in Phalangiacarus Coineau & Van der Hammen, and Naudo (1963), Coineau (1973), and Klompen (2000) noted the presence of distinct remnants of legs IV in larvae of, respectively, Panchaetes Naudo, Phalangiacarus, and Neocarus Chamberlin & Mulaik. Van der Hammen (1969) and Coineau and Van der Hammen (1979) also described the presence of sexually dimorphic tritonymphs in Neocarus platensis (Silvestri) and Phalangiacarus, and specified the addition pattern of the stigmata and sternitogenital setae over post-embryonic ontogeny. Most of these observations could be confirmed for specimens of Neocarus, Salfacarus Van der Hammen, Caribeacarus Va´zquez & Klompen, and a new genus from Australia available to us, with one exception: we have not been able to consistently recognize two forms of tritonymph (male and female) in most of the species we studied (Va´zquez and Klompen 2002, 2009, 2010), at least not using the characters identified by Van der Hammen (1969) or Coineau and Van der Hammen (1979). Specifically we have not noted a consistent difference in setation of the pregenital and genital region among putative male versus female tritonymphs in either of the four genera we studied for this phenomenon, suggesting that the phenomenon described for N. platensis and Phalangiacarus brossetti Coineau & Van der Hammen may be genus, or even species, specific. Interestingly, a number of Brazilian species appear to show sexual differentiation in adults and tritonymphs, but mostly expressed in the shape of the subcapitular setae (Bernardi et al. 2013b). The goal of this study is to document previously established and newly documented postembryonic changes in Opilioacarida, with the ultimate goal of using the combined information to establish inferences on homology and comparative development for all Parasitiformes.

123

Exp Appl Acarol

Materials and methods Material studied The new data presented are based primarily on specimens in three described genera, Neocarus, Salfacarus, and Caribeacarus, and undescribed material from Australia (description pending). Results were compared to available literature data for other genera. Specifically examined was material of Salfacarus antsirananensis Va´zquez & Klompen (all instars), S. ranobensis Va´zquez & Klompen (all nymphal instars, adults), Neocarus calakmulensis Va´zquez & Klompen (all instars), N. bajacalifornicus chamelaensis Va´zquez & Klompen (all nymphal instars, adults), N. texanus Chamberlin & Mulaik (prelarva, larva, adults), Caribeacarus armasi Va´zquez & Klompen (all nymphal instars, adults), and Phalangiacarus brossetti Coineau & Van der Hammen (adult male only), plus undescribed material (3 species) from Australia [all instars (across all 3 species)]. Individual specimens used for illustrations are listed by specimen code. Full collection data for this material is available at https://acarology.osu.edu. The general term ‘‘sensillum’’ is used in cases where the function of a given hair-like structure is unknown; ‘‘seta’’ is used only when the function of a hair-like structure is most likely mechanoreception only; ‘‘solenidion’’ is used for structures that externally resemble solenidia in Acariformes. The latter designation is tentative until TEM studies can reveal the internal structure of these sensilla. Designations for sensilla follow Grandjean (1936) with minor modifications (Va´zquez and Klompen 2002). Changes in developmental timing (heterochrony) are described using the terminology of Alberch et al. (1979). Finally, some specific comparisons are made with conditions in Acariformes. This does not imply support for a hypothesis of monophyly for ‘‘Acari’’ (we are neutral on this), it simply reflects the fact that data on early post-embryonic development in this group are more complete than in other arachnid groups.

Post-embryonic development in Opilioacarida Gnathosoma Chelicera (Fig. 1). Gross cheliceral morphology changes relatively little over development. The cheliceral base in the larva and protonymph appears more massive than in the following instars, resulting from a changed ratio of cheliceral to idiosomal size. The fixed digit in the larva is underdeveloped, distinctly smaller than the movable digit (Fig. 1a; also Naudo 1963), a difference that is no longer apparent by the protonymphal instar. While food remnants (pollen grains, mite cuticles, parts of insects) are easily seen in the gut of all post-larval instars (Va´zquez and Palacios-Vargas 1988; Walter and Proctor 1998), we have never observed food in the gut of opilioacarid larvae (N = 10, 5 species), suggesting they do not feed. All instars appear to have 2 lyrifissures (ia, id) on the fixed digit, although these structures (especially id) are often very difficult to find in the earlier instars. The number of cheliceral teeth, 1 on the fixed, 1–2 on the movable digit (not counting the terminal hook) is identical in the nymphs and adults of all species examined. Setation. In most species all instars have three setae on the fixed digit. Larvae, protonymphs and (most) deutonymphs lack setae on the basal segment with a single seta added in either the deutonymph (Caribeacarus, a few Salfacarus and Neocarus), tritonymph (most Salfacarus and Neocarus), or adult [Phalangiacarus (Coineau and Van der Hammen

123

Exp Appl Acarol

a

b ch1”

id cht

ch2”

iα

ch2’

c

Fig. 1 Chelicera, axial view. a Larva (Salfacarus antsirananensis Va´zquez & Klompen; OSAL 0007728), b male (Caribeacarus armasi Va´zquez & Klompen; OSAL 0007930), c female, detail ventral denticles on movable digit (S. antsirananensis; OSAL 0092012). b Redrawn from Va´zquez and Klompen (2009). Size bars a, b 100 lm; c 25 lm

1979)]. Exceptions to this general pattern are found in Paracarus which never adds setae to the basal segment, but appears to have four setae on the fixed digit (Van der Hammen 1968), as well as in males of Indiacarus Das & Bastawade, Siamacarus dalgeri Leclerc, and Caribeacarus armasi (Va´zquez and Klompen 2009) and males and some tritonymphs of three species of Brazilian Neocarus which carry 2–5 setae on the basal segment, and/or 5–20 setae on the fixed digit (Fig. 1b). Otherwise, cheliceral setation tends to be highly conserved. Observed teratologies are relatively rare, but include, for example, an extra seta on the basal segment. Ventral denticles (serrula) on the movable digit. The number of these structures in adults of Indiacarus and Salfacarus spp. can vary from 2 to 5 (with at least 1 quite large), while their number in Neocarus and Caribeacarus is generally limited to 1–2, rarely more. The number in Siamacarus is reported to be especially high, 8–10 in males (Leclerc 1989). In all taxa examined the number increases over ontogeny, starting with 1 (S. antsirananensis) or 0 (N. calakmulensis) in the larva. Palps (Fig. 2). Trochanter. Setal addition patterns on the palpal trochanter are highly consistent across taxa in the early instars. The palp trochanters in larvae and protonymphs never carry any setae, a single ribbed type seta [r-type of Va´zquez and Klompen (2009)] is added in the deutonymph, and additional setae (numbers may vary at this stage) are added in the tritonymphs and adults. Femur and genu (Fig. 2a–d). These segments show the usual addition of sensilla over development, but the pattern is much more variable than for the trochanter. In the larvae, all setae are of the ribbed type. One or two papilliform [p-type of Va´zquez and Klompen (2009)] setae appear on the genu of the protonymph in Salfacarus and the Australian species, but may be delayed until the tritonymph in some Neocarus and Caribeacarus. Tibia. Sensillar addition patterns for the tibia have not been studied. The number of setae on this segment is very large with some variability within instars. As such it was not considered informative for this study. Tarsus (Fig. 2e–j). Leaf-like (d) sensilla are absent in the larva. In most taxa examined, 2 leaf-like sensilla are present in the protonymph, 3 in the deutonymph, 4 in the tritonymph, and 5 or 6 in the adult. However, adults of P. brossetti, and Panchaetes papillosus

123

Exp Appl Acarol

a

b

c

r-type

d

p-type

g

e

i ch

sm

iπ

v v

f

d

h

s

d

j

ch sm

iα

s

Fig. 2 Palp. a, b Larva, whole palp, antiaxial and axial view (Neocarus calakmulensis Va´zquez & Klompen; OSAL 0007586). c, d, Palp trochanter to genu, combined axial (black) and antiaxial (white) view, c protonymph (Salfacarus antsirananensis Va´zquez & Klompen; OSAL 0007730), d deutonymph (S. antsirananensis; OSAL 0007736). Palp tarsus, antiaxial and axial view, e, f larva (N. calakmulensis; OSAL 0007586), g, h protonymph (S. ranobensis Va´zquez & Klompen; OSAL 0007293), i, j tritonymph (N. calakmulensis; OSAL 0007605). a, b, e, f, i, j Redrawn from Va´zquez and Klompen (2009), c, d redrawn from Va´zquez and Klompen (2010). Size bars a–d 100 lm; e–j 50 lm

(Andre´) have 7, and in Siamacarus Leclerc and Caribeacarus this number may reach 8–12. In contrast, in Neocarus orghidani Juvara-Bals & Baltac, N. nohbecanus Va´zquez & Klompen, a few undescribed species of Neocarus from Mexico and Brazil, and all Australian specimens the maximum number is 4. Among these exceptions, ontogeny has been studied in C. armasi, an undescribed Neocarus species from Mexico and one of the Australian species. In C. armasi protonymphs carry the standard number (2), while deutonymphs carry 4, and tritonymphs carry 6; in short, the deviation in sequence starts at the deutonymphal instar and takes the form of acceleration. In the Australian species addition proceeds normally until the tritonymph, but this species lacks the final (adult) addition:

123

Exp Appl Acarol

paedomorphosis in the form of progenesis. Most deuto- and trito-nymphs of the Mexican species show the same pattern, but one deutonymph carried only 2 d sensilla, and one tritonymph carried only 3, consistent with either neoteny or post-displacement. More research will be required to clarify this unexpected diversity in inferred processes. Notably, even when additional d sensilla are present, the post-embryonic addition sequence within a species appears to be constant, with a possible exception of values in the adults. Post-adult molts occur in Opilioacarida (Bernardi et al. 2013a; Coineau and Legendre 1975; Klompen 2000) and may affect some sensillar numbers (e.g. in the ‘‘supermales’’ noted in Va´zquez and Klompen (2002)), but not the number of d sensilla. All known larvae carry two s sensilla (solenidia?), a third is added in the protonymph in Salfacarus and C. armasi, or in the deutonymph for N. calakmulensis. There are no further additions. The numbers for the v, ch, and sm sensilla increase over ontogeny, but not in a consistent manner. Two lyrifissures, ia and ip, are present in all postlarval instars. The condition in the larvae is unclear in the specimens available. Pretarsus. The well-developed pretarsal claws are unique to Opilioacarida. They are homologous to the ‘‘palpal apotele’’ in Mesostigmata and Holothyrida (Camin 1958). We have not observed significant changes in these structures across ontogeny. Subcapitulum (Fig. 3). The paralabial sensilla in opilioacarid larvae are poorly developed. With’s organ (pl2) is small and cone-like and the rutellum (pl3) has no or only weakly developed teeth (Fig. 3a; Naudo 1963). Both of these structures are well developed in all post-larval instars (Fig. 3b). The number of circumbuccal (cb) setae increases from 2 in the larva to 4 or 5 in the post-larval instars. The presence of a 6th circumbuccal-style seta characterizes females and female tritonymphs (vs. males and male tritonymphs) in some Brazilian taxa (Bernardi et al. 2013b). Circumbuccal-style setae are characterized by thick, blunt or bifurcate tips, which differentiate them from the median and subcapitular setae [vm,

b

a

pl3

pl3

pl2

pl2 pl1 cb cb

cb

cb pl1

Fig. 3 Subcapitulum, ventral view. a Larva (Salfacarus antsirananensis Va´zquez & Klompen; OSAL 0007728); b deutonymph (Neocarus calakmulensis Va´zquez & Klompen; OSAL 0007594). cb circumbuccal sensilla, pl1-3 paralabial sensilla, pl2 With’s organ, pl3 rutellum. Sensillum pl4 inserted dorsal (not figured). Size bar 100 lm

123

Exp Appl Acarol

a

b ly

VIII/IX X XI VI

XII

VII VIII

XIII

g sv

IX

jv

zv

XIV XV

X

XVI XVII

XI

ly

XII XIII

j

z

XIV

XVIII

s

XV XVI

c

XVII

XVIII

d

sv ly

VI VII

gc VIII/IX ?

X XI XII

?

XIII ?

XIV

? ?

XV XVI XVII

XVIII

Fig. 4 Idiosoma, larva, reconstructed pattern of idiosomal setae, lyrifissures and glands. Putative segmental homology—indicated by Roman numbers. a, b Neocarus calakmulensis Va´zquez & Klompen (OSAL 0007586); c, d Salfacarus antsirananensis Va´zquez & Klompen (OSAL 0007728). a, c dorsum; b, d venter. g gland, gc genital capsule, ly lyrifissure, sv sternal verrucae. Presumed ‘‘missing’’ lyrifissures indicated by question marks. Size bar 200 lm

lvm, ldm, vp, lvp of Van der Hammen (1966)], which have thin, attenuate, tips. Median and subcapitular setae are first added in the protonymph. Their number increases over development, but is variable within instars (esp. in adults). The weak development of the subcapitular structures in larvae is consistent with the hypothesis that larvae may not feed.

123

Exp Appl Acarol

a

b

ly

sv ly VI

gc

g VII

VIII/IX

ly X

VIII/IX XI X

st

XII

XI XIII XII

XIV

XIII

XV

XIV

XVI

XV

XVII

XVI

XVIII

XVII

XVIII

c

d

sv VI VII VIII IX

gc

IX

X

X

XI XI XII XIII XIV XV

XII

j

z

s

r

XIII XIV XV XVI

XVI XVII

XVIII

123

XVII

XVIII

jv

zv sv rv

Exp Appl Acarol b Fig. 5 Idiosoma, reconstructed pattern of idiosomal setae, lyrifissures and glands. Putative segmental homology—indicated by roman numbers. a, b Salfacarus ranobensis Va´zquez & Klompen, protonymph (OSAL 0007290, 0007297); c, d S. ranobensis, deutonymph (OSAL 0007311, 0007312). g gland, gc genital capsule, ly lyrifissure, st stigma, sv sternal verrucae. c, d only: open circles: setal bases; grey lyrifissures: observed in only one specimen (others observed in both). Size bar 200 lm

Idiosoma (Figs. 4–6) Segmentation. One commonly noted characteristic in which Opilioacarida differ from other Parasitiformes is that the posterior idiosoma shows distinct signs of external segmentation. The other Parasitiformes lack such clear external indications, although the festoons in some Ixodida (ticks) have been suggested as segmental indicators (Klompen et al. 1996; Schulze 1932). In contrast, studies on possible segmentation patterns in Mesostigmata have relied on sensillar patterns (e.g. Athias-Henriot 1971). In a signature study Van der Hammen (1966) examined adults of Neocarus texanus and, based on the rough pattern of body constrictions, muscle attachments (sigilla), and lyrifissures, proposed the presence of 20 segments, starting with the chelicera. This included an opisthosoma with 14 segments, 2 of which formed the anal cone. He later modified this model suggesting that the anal cone included only a single segment (Van der Hammen 1969, 1970). The model included the notion that the dorsal expression areas of the pregenital and genital segments are fused, and that the dorsal elements of the segments associated with legs III–IV are expressed solely in the posterior section of the anterior dorsal shield. Sitnikova (1978) re-interpreted Van der Hammen’s observations, agreeing that the pregenital and genital segments were fused dorsally, but suggesting that the dorsal sections of the segments of legs III–IV were not fused, and rejecting/omitting one opisthosomal segment. The end result was a total of 17 segments (starting with the chelicera and recognizing only 1 segment for the anal cone). The current study allows testing of some elements of these hypotheses based on an explicit consideration of developmental patterns.

Epimorphosis versus anamorphosis and the number of segments Propodosoma. The body of most Arachnida consists of a cephalothorax or prosoma (including the mouthparts and legs I–IV), and an abdomen or opisthosoma, although this division is obscured in Acari. The body of many Acariformes shows a distinct division into a propodosoma (all segments up to, and including, legs II) and a hysterosoma (the area posterior to legs II; Krantz 2009). The hysterosoma itself can be divided into the metapodosomal region (the area including legs III–IV and corresponding dorsal areas) and the region posterior to the legs (opisthosoma). Even though these three regions are not distinct in Parasitiformes, they are discussed separately for practical regions. Analyses of developmental genetics (Damen et al. 1998; Telford and Thomas 1998) support the notion that the propodosoma in Arachnida, including Acari, consists of segments associated with, respectively, the eyes, chelicera, palps, and two leg segments, for a total of 5. The ‘‘segment’’ including the eyes may not be quite similar to other segments in structure and origin, reflected in its alternate designations as the ocular or protocerebral region (Scholtz and Edgecombe 2006). We acknowledge these concerns, but for ease of discussion we will use the term ocular segment. Whether or not there is an acron or a separate labral region, in addition to the above-listed set, is not relevant to this discussion (see Scholtz and Edgecombe 2006). The implications of the presence of an ocular segment

123

Exp Appl Acarol

have not always been fully appreciated. If all eyes are positioned on that segment and do not move between segments (similar conditions as generally accepted in insects), then the ocular segment in all Arachnida should include both median and lateral eyes. This means that most of the anterior dorsal shield in Opilioacarida, and the scutum in immature and female Ixodidae would represent the ocular segment (the condition in Mesostigmata, which never have eyes, is less obvious, but see below). Under this hypothesis, it is likely that there is little or no dorsal expression of the segments associated with the cheliceral or palpal segments, or of the segments of legs I–II (some lateral expression cannot be excluded) as (nearly) all of the dorsal propodosoma is made up of the ocular segment. This is consistent with earlier hypotheses, be it that those were developed for Acariformes. Grandjean (1954, 1969) recognized a pre-cheliceral entity (his ‘‘ACa’’), and proposed that idiosomal representation of the pre-cheliceral, cheliceral and palpal segments was in the form of the ‘‘aspidosoma’’, the prodorsal region antero-dorsal to the abjugal furrow. He also concluded that the segments associated with legs I–II had no dorsal expression. This Acariform model seems to fit our observations for Opilioacarida with a few differences: (1) Grandjean did not associate this view with distribution of eyes, as suggested above; (2) our hypothesis rejects dorsal expression of the cheliceral and palpal segments in the dorsal region of the idiosoma (all ocular segment in our view). Given this complexity, we will not attempt to hypothesize segmental borders in this region of the body, and restrict ourselves to the observation that the propodosoma in Opilioacarida includes elements of at least five segments, the ocular, cheliceral, palpal, leg I and leg II segments. The region posterior to legs IV. Segmentation of the region posterior to the dorsal shield of Opilioacarida is relatively straightforward. Larvae, protonymphs, and deutonymphs of all Neocarus and Salfacarus spp. studied (Figs. 4, 5) are characterized by the same remarkably regular pattern of lyrifissures, glands, and, occasionally, setae, identified earlier in larvae of N. texanus (Klompen 2000). Study of additional specimens allowed more definitive statements on regions that were unclear in that earlier study. The opisthogaster of all larvae, protonymphs and deutonymphs includes 8 distinct rows of lyrifissures and glands (Figs. 4b, d, 5b, d). Assuming that each represents a segment, with additional segments for the genital region (pregenital and genital segments) and the anal lobe, this suggests that the opisthosoma of larval and protonymphal Opilioacarida has at most 11 segments. The dorsal pattern is not quite as simple. In larvae and protonymphs it includes 11 (Neocarus; Fig. 4a) or 10 (Salfacarus; Fig. 4c) rows of lyrifissures and glands posterior to the dorsal shield, plus the anal lobe. It seems unlikely that different genera within a morphologically homogeneous group such as Opilioacarida have different numbers of primary segments, so we suggest that Neocarus has separate dorsal rows of lyrifissures and glands for the pregenital and genital segments, while these are combined in one row in Salfacarus. A similar assumption was made by Klompen et al. (1996) to accommodate added setae and small glands in some ixodid larvae, and to accommodate added setae and glands in ixodid nymphs versus larvae. This hypothesis is consistent with the observation that deutonymphs of Salfacarus appear to have the full complement (11) of rows or lyrifissures and glands (Fig. 5c). Lyrifissure and gland patterns in tritonymphs and adults are more difficult to interpret due to extensive hypertrichy, but a study of external segmentation and muscle attachment patterns in these instars (following the methods of Van der Hammen (1966), Figs. 1–3) suggests that these instars have the same number of rows as the deutonymphs (see Fig. 6). The latter result is again largely consistent with literature observations. Grandjean (1936) for Opilioacarus segmentatus With, Naudo (1963) for Panchaetes dundoensis Naudo, and Van der Hammen (1966) for N. texanus, all noted 11

123

Exp Appl Acarol

pre-gen gen X XI XII IX legIII leg IV VIII VI

VII

XIII

XIV

XV

XVI XVII

I-V

e

XVIII

s

o

Fig. 6 Idiosoma female Salfacarus kirindiensis Va´zquez & Klompen, lateral view showing external segmentation. Putative segmental homology—indicated by roman numbers. Legs and palps not shown; setation incomplete. e eye, gen genital, o ovipositor, pre-gen pre-genital, s stigma. Size bar 200 lm

segments/transverse series of setae/lyrifissures/muscle attachments in adults between the dorsal shield and the anus. Sitnikova’s (1978) re-interpretation of Van der Hammen’s study suggesting only 10 transverse series is inconsistent with available data. Together these observations suggest that (1) combination of the pregenital and genital segments may have an ontogenetic component, and (2) there is no addition of opisthosomal segments in the development of Opilioacarida, in other words development is epimorphic. We found one additional discrepancy with previous results. Van der Hammen (1966) noted 10 transverse series of lyrifissures/muscle attachments in adults of N. texanus ventrally between the genital segment and the anus. We have not been able to replicate that observation in any of our material. As noted above, the number we can distinguish between the genital region and the anal cone in a variety of instars and in a variety of genera is consistently 8. Unlike Van der Hammen, we therefore recognize only 11, not 13, opisthosomal segments. The region associated with legs III–IV. As noted above, the dorsal area posterior to the dorsal shield includes 11 rows of lyrifissures and glands, plus the anal cone, for 12 putative segments. Comparing this with observations for the opisthosomal venter (11 putative segments), leaves the anterior row of dorsal lyrifissures and small glands unassigned. Second, in larvae and protonymphs (Figs. 4a, c, 5a) there is a remarkable continuity between this row of lyrifissures and small glands and a row of setae and small glands on the posterior edge of the dorsal shield (labeled as segments VI and VII in Figs. 4, 5). Serial homology of these outwardly highly dissimilar structures, setae and lyrifissures, is based on the notion that they are structurally and functionally similar. Both setae and lyrifissures are mechanoreceptors with similar innervation (Alberti and Coons 1999; Altner and Prillinger 1980; Evans 1992), differing by the presence or absence of a hair-like portion. As such they are very different from typical glands which lack innervation. We hypothesize that both rows (one with setae, one with lyrifissures) represent segments, specifically the segments associated with legs III and IV (segments VI and VII in our system). The hypothesis that elements of the dorsal shield could represent dorsal expression areas of leg segments follows Van der Hammen (1970), while the hypothesis that dorsal expression of

123

Exp Appl Acarol

the segments of legs III–IV might span areas of both shield and non-shield appears implicit in Sitnikova’s (1978) hypotheses. As an aside, the above hypothesis would render moot the discussion on whether the area posterior to the dorsal shield in Opilioacarida is equivalent to the hysterosoma or the opisthosoma: it is neither. Based on the above hypotheses, the total number of primary body segments of opilioacarid mites is thus assumed to be 18: ocular, cheliceral, palpal, 4 leg segments (segments IV–VII), and 11 opisthosomal segments. The latter include the pregenital and genital segments (VIII, IX), 8 additional opisthosomal segments (X–XVII), and a segment representing the anal valves (XVIII). Given this, segmental homologies for stigmata can be updated as follows. The stigmata in the tritonymphs and adults are positioned on segments VIII, IX, X, and XI (designated as stigmata 1–4). They are added in the following sequence: no stigmata in the larva (as in all Parasitiformes), stigmata 2, 3 added in the protonymph, 4 added in the deutonymph, 1 added in the tritonymph (Coineau and Van der Hammen 1979). The statement by Chamberlin and Mulaik (1942) that stigmata 2–4 are all on the second ‘‘abdominal’’ segment is incorrect, each pair is inserted on a separate segment (Van der Hammen 1966). This model does differ from both Van der Hammen (1966, 1970) and Sitnikova (1978) in recognizing dorsal expression of the pregenital segment (VIII). Otherwise Van der Hammen’s results (and drawings of N. texanus) are largely compatible with our interpretation (Fig. 6). Chaetotaxy and porotaxy. As noted above, the current study confirmed the existence of a strikingly consistent pattern of lyrifissures and small glands on the idiosoma of the larvae (Klompen 2000). In larvae of Neocarus, Salfacarus and the Australian species we found rows of six (rarely four) pairs of lyrifissures, and four pairs of small glands on both the dorsum and the venter. In discussing this pattern we will use the modified Sellnick terminology of Lindquist and Evans (1965). This system was developed for Mesostigmata. In that group it is possible to consistently recognize transverse rows of up to eight setae on the dorsum of post-larval instars. Given bilateral symmetry, these have been designated as pairs of setae arranged in parallel series, from inside to outside the J (or I), Z, S, and R series. Interpretation of ventral, specifically opisthogastral, setae is less straightforward. The anterior movement of the anus (from a presumed ancestral terminal position still observed in Opilioacarida) resulted in compression of segments in the opisthogaster, and a strong reduction of the number of setae. As a result setal designations in this area are tentative. Even so, setae have been assigned to the Jv, Zv, Sv, or UR series (Lindquist 1994; Lindquist and Evans 1965). Of these, the R and UR (or Rv see below) setae are never present in mesostigmatid larvae; if added at all, they appear in the post-larval instars. Since its inception this system has been extended in various ways. Even though much of the thinking leading up to this system was based on earlier work by Athias-Henriot (1957), which focused heavily on the idea that setal rows might reflect ancestral segmentation, the authors (Lindquist and Evans 1965) specifically noted that their system was not necessarily a hypothesis of segmental homology. We believe that there is sufficient evidence to support such a view, at least for the hysterosoma/opisthosoma (Lindquist 1984). Moreover, although developed for Mesostigmata, we have successfully applied this system to other parasitiform suborders, including larval Ixodidae (Klompen et al. 1996), Opilioacarida (Klompen 2000), and Holothyrida (Klompen 2010). The distribution pattern of cuticular structures (setae, lyrifissures, glands) in larval Opilioacarida is particularly striking in this context, as it appears to fit the model better than any of the mesostigmatid species for which it was designed. Not only is the dorsal pattern of lyrifissures/setae in the area

123

Exp Appl Acarol

posterior to the dorsal shield nearly complete, so is the ventral one (Fig. 4b, d). The one caveat is that ‘‘complete’’ in this case is complete for larvae: the R/UR series are absent. Given that caveat, it is relatively easy to assign all visible lyrifissures/setae to one of the six sensillar series, with very few positions ‘‘unoccupied’’. Segmentally arranged small glands are found between the J and Z, and the Z and S lyrifissures on the dorsum, and between the Jv and Zv, and the Sv and S lyrifissures on the venter. A study of patterns in nymphal instars suggests that the larval pattern is maintained in the protonymph (Fig. 5a, b), but in the deutonymph additional rows of lyrifissures get added on both dorsal and ventral side, apparently lateral to existing sensilla (Fig. 5c, d). These lyrifissures are hypothesized to be equivalent to the R and UR series in Mesostigmata. As noted by Lindquist (1994) the ventrolateral UR series belongs to the ventral chaetome, and based on the evidence presented here, we support Lindquist’ (1994) suggestion to use Rv, rather than UR, as the preferred setal designation, to retain consistency with the other elements of the system. We note these observations with some reservations: discovery of individual lyrifissures/setae/glands gets progressively more difficult in deutonymphs and subsequent instars, and even study of multiple individuals could not establish complete patterns. In the tritonymphs and adults (and some deutonymphs) supernumerary structures start appearing, rapidly obscuring recognition of individual sensilla or glands. In summary, larvae and protonymphs in Opilioacarida show a pattern of segmentally arranged, designatable sensilla (lyrifissures and setae) in the area posterior to the dorsal shield and, on the ventral side, posterior to the genital region. This pattern includes near complete series of J, Z, S, Sv, Zv, and Jv sensilla. R and Rv series sensilla are added in the deutonymph. Supernumerary sensilla in the tritonymphs and adults retain segmental association (e.g. Van der Hammen 1966), but can no longer be designated individually. In most genera the added structures in development are lyrifissures and glands, but in Panchaetes, Salfacarus, Vanderhammenacarus Leclerc, and some species from Australia and Brazil varying numbers of setae are added in the region posterior to the shield in the adults. These additions start in the deutonymph, with additional setae added in the tritonymph and adults. The initial additions are restricted to the mid-dorsal region between the dorsal Z series of lyrifissures, and mid-ventral region between the Zv series of lyrifissures. The absence of setae in the larva and protonymph, and the irregular pattern of setae in the post-protonymphal instars (in contrast to the regular pattern of lyrifissures) suggest that the setal addition is secondary, while the lyrifissure pattern is primary. Finally, the character of having only three setae on the pre-anal segment in all instars, one dorsal, two ventrolateral (diagnostic for Neocarus and Caribeacarus) results from lack of addition of setae. Most described Old World genera (Opilioacarus, Phalangiacarus, Panchaetus, Indiacarus, Salfacarus), as well as the Australian species and a few taxa in Brazil appear to start with the same complement, but add setae during post-embryonic development. The condition in Adenacarus With and Paracarus Chamberlin & Mulaik (no pre-anal setae) needs more study. Prodorsal shield region. All instars in all Opilioacarida have a similar shaped and sized prodorsal shield. It is weakly sclerotized, and contains 2–3 pairs of lateral eyes (absent in Siamacarus dalgeri Leclerc), one pair of lyrifissures (both eyes and lyrifissures are invariant in number across instars), a few small glands, and a number of setae. The number of setae increases over development, but numbers are highly variable within instars, especially tritonymphs and adults. With few exceptions setae are monomorphic.

123

Exp Appl Acarol

Sternitogenital region. The addition sequence of setae in the sternitogenital region again shows some striking similarities with that observed for Mesostigmata, at least in the early instars. The larvae carry one pair of setae on the sternal verrucae (sv), and two pairs on the main sternal region. They lack setae on the genital capsules (gc in Fig. 4b, d). In the protonymph (Fig. 5b) they add one pair of setae on the genital capsules. This addition sequence resembles that observed in Mesostigmata, and we propose designating these setae as St1 (sternal verrucae), St2 and St3 (sternum proper) and St5 or g (genital capsule). Hypertrichy appears starting in the deutonymphal instar, but the St setae can often still be recognized because of their slightly different shape (Bernardi et al. 2013b, c). Anal valves. The anal plates start with two setae each in the larva, a pattern maintained in the protonymph. From the deutonymph on the pattern becomes irregular. Adults may carry more than 20 setae on each anal plate.

Legs Segmentation. The basic segmentation of the opilioacarid legs is constant, with a coxa, trochanter, femur, genu (or patella), tibia, tarsus and pretarsus, but secondary divisions may occur on the tarsus, tibia, femur, and trochanter. The tarsus on all legs is divided into a basi- and a telotarsus, although basitarsus I is poorly differentiated from the telotarsus (Grandjean 1936). On legs II–IV, the distal part of the telotarsus may also be separated as an acrotarsus. Only the acrotarsal differentiation has an ontogenetic component. The acrotarsus on legs IV appears in the deutonymph of most species, but appearance of distinct acrotarsi on legs II–III is usually delayed to the tritonymph. Subdivision of the tibia is restricted to tibia I. It may be very distinct, as in Opilioacarus and Panchaetes, or weakly developed or absent as in Caribeacarus, Indiacarus, Neocarus, Paracarus, Salfacarus, and the Australian species. When present, the division appears at the tritonymphal instar. A secondary division of the femur into a small basifemur and long telofemur is found in nearly all Parasitiformes (Lindquist 1984). In opilioacarid adults, this division is well developed for legs I, but remarkably poorly developed for legs II–IV. Finally, a highly unusual (for Parasitiformes) developmental pattern is seen in the apparent split of the trochanters of legs III–IV. Trochanters IV may be split starting in the deutonymph (some Neocarus and Salfacarus), or in the tritonymph (Caribeacarus, most Neocarus, Phalangiacarus, Panchaetus, some Salfacarus), trochanters III are split one instar later. There are differences of opinion on whether the split involves two trochanters (Van der Hammen 1966, 1989) or a version of the common split between telo- and basifemur. Naudo (1963) suggested the latter, based on similarities in cuticular patterning of the femur and distal ‘‘trochanter’’, a view supported by Shultz (1989) based on a comparative study of leg musculature across Arachnida. Notably, even, Coineau and Van der Hammen (1979) refer to trochanter 2 as ‘‘incorporated in the femur’’ in early instars. As a counter argument, it is unclear what the poorly developed secondary division of femora III–IV represents under this hypothesis. Solenidia. A number of sensilla that externally resemble solenidia are present on the legs of Opilioacarida. Most are present on tarsus I but given the exceedingly complex structure of that segment (Moraza 2005), we will concentrate on the remaining leg segments. Acrotarsus II carries two solenidia, one longer and distal, the other short and basal (the latter is displaced onto the telotarsus in Phalangiacarus and a few undescribed Brazilian species). This appears to be constant throughout ontogeny. The basitarsi also carry two

123

Exp Appl Acarol

a

b

c

d p r

g ωd r

coronidia

ud

e

ωp

ωd

pld pl plv ald

ibt

al alv av pv

ibv

Fig. 7 Basitarsus and tibia of legs III (a, b) and IV (c–e). a, b Salfacarus antsirananensis Va´zquez & Klompen, c–e Salfacarus ranobensis Va´zquez & Klompen. a Larva (OSAL 0007728), b protonymph (OSAL 0007729), c protonymph (OSAL 0007287) with details of sensillar shape, d deutonymph (OSAL 0007312), e tritonymph (OSAL 0007318). al ald alv av pl pld plv pv hypothesized designations for paired sensilla, bTa basitarsus, g gland, ibt ibv lyrifissures, xd xp solenidia, p fan-shaped papillate seta, r ribbed, tapering seta, Ti tibia, ud unpaired dorsal row of sensilla. Size bars a–d 100 lm, e 200 lm

solenidia, one dorsal, one lateral. On legs I, both are inserted in the middle of the segment, but on basitarsi II-IV, the dorsal one is basal (xp), the lateral one distal (xd). Solenidion xd may be positioned in a cavity (Fig. 7c), although this can be variable across ontogeny and phylogeny. In Neocarus xd appears normal in the larva and protonymph, but it is in a

123

Exp Appl Acarol

cavity starting with the deutonymph, while xd appears in a cavity in all nymphal instars in Salfacarus and the Australian specimens. Solenidion xd is also in a cavity in adult Phalangiacarus. Data on other genera is currently lacking. Finally, in some species additional solenidia may be present on tibiae I (Van der Hammen 1966), starting with the tritonymph, and with numbers increasing in the adult (Bernardi et al. 2013b). These solenidia are often very difficult to see, and their distribution across genera is unclear. Setae. Setation of the legs does not follow standard mesostigmatid patterns with one or more whorls of up to six setae per segment. Variability appears in (1) the presence or absence of unpaired dorsal sensilla on the basitarsi, tibiae, genua, and femora, (2) the shape of the leg sensilla, (3) the number of whorls per segment, and (4) the number of setae per whorl (ignoring unpaired dorsal setae). (1) The most common unpaired sensilla on the basitarsi are thin, smooth, and curved [the ‘‘coronidia’’ of Van der Hammen (1977); Fig. 7e]. They are generally inserted relatively basal in position. Later in ontogeny (starting at the deutonymph), a second type of unpaired sensilla, ribbed, tapering sensilla may be added, always distal to the coronidia (Fig. 7d: r). The Australian species never add ribbed tapering sensilla. The total number of unpaired sensilla on the basitarsi increases over ontogeny from 1–4 in the protonymph to 13–22 in the adults, but numbers appear somewhat variable both among and within species. In a second trend, the numbers on basitarsi IV tend to be higher than those on basitarsi II-III, a difference maintained in all instars. On the tibiae the most prominent structure is a distal, mid-dorsal, unpaired and elongate sensillum, sometimes with a thin, pointed extension (detail in Fig. 7c). This sensillum is longer than any other sensillum on that segment, and it is present in all instars. Additional unpaired sensilla may be added on tibiae IV in the protonymph (Neocarus, Salfacarus) or deutonymph (Caribeacarus). Such sensilla on tibiae II–III are generally added one instar later than on tibiae IV. Numbers increase from 1 (rarely 2) extra when first added, up to 9 in some Neocarus and Salfacarus. Numbers for Caribeacarus and Phalangiacarus are distinctly lower (3–6). Nearly all of these additional sensilla, and any unpaired sensilla on the genua or femora are of the ribbed, tapering type. Coronidia are added on the tibia and even genu in Panchaetes (Van der Hammen 1977) and a few species of Brazilian Neocarus, but not in the other taxa examined. Unpaired sensilla on the genua appear first in the protonymph (Caribeacarus, Salfacarus) or deutonymph, once again first on genua IV, than on genua II–III. Interestingly, we found no unpaired sensilla on the genua of the one adult Phalangiacarus (a male) available for study. Patterns on the femora are more difficult to interpret due to frequent lack of symmetry, but unpaired sensilla appear to be delayed until the deuto- or tritonymph. Numbers of unpaired sensilla on the genua or femora are highly variable, more so than for the basitarsi or tibiae. (2) Leg setae in the larvae are mostly of the ribbed, tapering type (Fig. 7c: r). In later instars some setae get changed to broader, more fan-shaped setae, occasionally with a tiny terminal tip (‘‘papillate’’ type; Fig. 7c: p). Although these ‘‘replacements’’ are most common for dorsal setae, they are not uniform, and patterns of replacement vary considerably among genera and species within genera. (3) The number of setal whorls appears to be relatively constant for acrotarsi and telotarsi II–IV, with respectively 3 and 6 whorls, although all Caribeacarus and some Neocarus carry 4–5 whorls on the acrotarsi. The number of whorls on all other segments studied clearly increases over ontogeny, possibly to accommodate coverage of a larger sensory area (Klompen 2000). The basitarsi, tibiae and genua in larvae and protonymphs

123

Exp Appl Acarol

carry 3 whorls each, the femora may carry 4 (femora II–III) or 5 (femora IV). These numbers for the four segments increase to, respectively, 8–11, 6–13, 5–10, and 8–20 in the adults. Across the taxa studied, numbers tend to be highest for Neocarus, lowest for Phalangiacarus. (4) The number of setae per whorl also changes over ontogeny. Unlike larval Allothyridae (Holothyrida) (Klompen 1992), but similar to larval Neothyridae (Holothyrida) (Klompen 2010), Ixodida and all instars of Mesostigmata, opilioacarid larvae, protonymphs and deutonymphs show whorls of at most six setae (the condition for tarsus I is not considered). This condition is maintained for the telotarsi and (most) basitarsi, but starting with the tritonymph, the tibiae, genua, and femora consistently show whorls of eight setae (Fig. 7e). It is unclear where the ‘‘extra’’ setae are added (dorsal, lateral or ventral). It is possible that this change also has a functional aspect, with more setae required to cover the much larger surface area of the leg segments in older instars.

Comparison with other Parasitiformes General fit of the model A comparison of the above Opilioacarid model of segmental arrangement with existing models of segmentation in mites reveals a strong similarity with the generalized acariform model proposed by Grandjean (1969). This model has two components of interest. First the presence of an ‘‘aspidosoma’’ on the anterior dorsum of the idiosoma, delimited laterally by the abjugal furrow, and dorsally by the sejugal furrow. Although the furrows as such are not present in Opilioacarida, we propose that the segmental arrangement in the propodosomal region is similar. The second component includes a dorsal ‘‘extension’’ of the opisthosomal segments up to the sejugal furrow, limiting, or even blocking out, dorsal expression of the segments of legs III–IV. In Acariformes, this dorsal extension is delimited laterally by the disjugal furrow. We propose that the latter component is also shared with Opilioacarida (and other Parasitiformes; see below). However, unlike the condition for segments I–V, there is less structural need for this modification and, as noted above, Opilioacarida do retain some dorsal expression of the segments of legs III and IV (segments VI and VII in our model). The Grandjean model has been criticized for being speculative (Alberti and Coons 1999; Evans 1992) and unnecessarily complicated (Shultz 2007), and far simpler models have been proposed (e.g. Weigmann 2001). However, these alternative models do not accommodate the ocular segment and/or do not consider eyes as part of the ocular segment. For that reason we do not consider these models appropriate. Moreover, as noted below, a single process can be invoked that will generate nearly all proposed segmental ‘‘contortions’’, resulting in a relatively simple hypothesis with considerable explanatory power. A different possible criticism, using the absence of distinct abjugal, sejugal, and disjugal furrows in many mites, such as Opilioacarida, as de facto evidence against the Grandjean hypothesis also seems unconvincing. After all, few would reject the notion that Parasitiformes are segmented animals even if external segmentation is commonly obscured. It is worth noting that although the most common interpretation of this pattern assumes an anterior movement of the dorsal opisthosomal segments (e.g. Van der Hammen 1989; Weigmann 2001), this is not required, and may not even be the most parsimonious explanation. The alternative is based on a hypothesis of a similar type of differential

123

Exp Appl Acarol

growth in all appendage-bearing segments (II–VII): the coxal region grows strongly, while growth in the dorsal (and mid-ventral) region is very limited or absent (Fig. 8a, b). This single process would simultaneously result in the dorsal (rather than anterior) position of the ocular segment (component one of the Grandjean hypothesis), the antero-ventral position of the gnathosoma, as well as the dorsal and ventral ‘‘extensions’’ of the opisthosoma (component two). The ventral extension is rarely emphasized (and generally less pronounced), but the common presence of the genital region (fundamentally opisthosomal) between coxae III–IV requires explanation. Strong growth in the coxal area of legs III–IV, combined with little or no growth in the dorsal and mid-ventral areas of segments VI–VII would result in the coxae pushing into the opisthosoma, resulting in the apparent ‘‘extensions’’ of the dorsum and mid-ventral regions. Presumably differential growth within segments also has to be invoked for the posterior opisthosomal segments to

a

V

III I II

IV

VII IX XI XIII XV XVII VI VIII X XII XIV XVI XVIII

ocu pal LII LIV gen che LI LIII pre

b

VIII

anal

IX

X

XI

XII

XIII

XIV XV

I

XVI II XVII III

XVIII IV V

c

VI VII VIII/IX

X

XI

XII

XIII

I

XIV

II XV III IV V

VI VII

XVIII

XVI XVII

Fig. 8 Schematic representation of the segmental model proposed. Putative segmental homology— indicated by roman numbers. a Hypothetical isomorphic ancestor. Filled dark circles indicate appendages, filled light circle eye(s). b Model for Opilioacarida resulting from strong growth in the coxal regions of segments II–VII and slow or no growth in the dorsal regions of those segments (arrows indicate direction of resulting segmental rearrangements). c Possible model in Mesostigmata: similar to Opilioacaridae, but dorsal expression of segments VI and VII absent and dorsal regions of segments VIII and IX fused. Differential growth in the posterior segments results in a ventral, rather than terminal position of the anus. For the purpose of this model the border between segments V and VI (legs II and III) is kept constant. che cheliceral, gen genital segment, LI-IV legs I–IV, ocu ocular, pal palpal, pre pregenital

123

Exp Appl Acarol

explain the caudal bend and ventral position of the anus in Ixodida and Mesostigmata (assuming a terminal anus, as in Opilioacarida, is primitive; Fig. 8c). Finally, the same model of differential lateral growth proposed above for podal segments, if slightly redirected to accelerated dorso-lateral (rather than ventro-lateral) growth in the opisthosomal segments, would result in the segmental distortion pattern shown in Fig. 9 for the opisthosoma of ixodid larvae, including the laterally compressed segment XVII on the dorsum, the minimal growth of the mid-dorsal and mid-ventral elements of segments X– XVI, and the caudal bend. The match of the results of the current study with data from embryology or developmental genetics is mixed. For example, morphological studies of tick embryos (e.g. Aeschlimann and Hess 1984) recognize at most five opisthosomal segments. A partial explanation of this contradiction may lie in the observation that it is not unusual for some of the terminal opisthosomal segments to form quite late in embryonic development in Arachnida (e.g. Wolff and Hilbrant 2011), and it is unclear whether the above study followed embryogenesis until hatching. In this context, it is worth noting that molecular embryology of the tick Rhipicephalus (Boophilus) microplus (Canestrini) showed indicators of at least eight opisthosomal segments based on DAPI and antennapedia (Antp) antibody staining (Santos et al. 2013).

a

b I ly

IV V

VI g VII VIII/IX ly

X XI XII XIII XIV XV XVII

XVI

XIII lwg

XIV XV XVI

XVII

Fig. 9 Ixodidae: Amblyomma americanum (Linnaeus), larva, idiosoma. a Dorsum, b venter. Putative segmental homology—indicated by roman numbers; proposed segmental borders indicated by dashed lines. Redrawn and modified from Klompen et al. (1996). g gland, lwg large wax gland, ly lyrifissure. Size bar 200 lm

123

Exp Appl Acarol

Fit with other Parasitiformes Previous studies (Alberti and Coons 1999; Lindquist 1984) have largely followed Van der Hammen (1966) hypothesis that the ‘‘true’’ Parasitiformes (Mesostigmata, Ixodida, Holothyrida) have fewer fundamental segments than Opilioacarida. This may be inaccurate, as it is quite possible to construct plausible models of segmentation for ixodid ticks and Mesostigmata (very little is known for holothyrids or argasid ticks) if we assume (1) the opilioacarid model of limited dorsal expression of segments II–VII holds (likely), (2) the anterior dorsal ‘‘extension’’ of the opisthosomal segments is as extensive as in Opilioacarida, and (3) the pre-genital and genital segments are fused dorsally [an assumption made by Sitnikova (1978) and Van der Hammen (1966), and inferred for larval Salfacarus (Fig. 4c)]. In the following we will restrict ourselves to the dorsum, as the strong reduction in setation on the opisthogaster of Ixodidae and Mesostigmata disallows substantiated hypotheses of homology for that region. If all Parasitiformes have 18 primary segments, larval Ixodidae can be accommodated as follows: the five pairs of festoons of metastriate Ixodidae represent segments XII–XVI [not IX–XIII as proposed by Klompen et al. (1996)], with the parma (the unpaired central festoon) representing segment XVII, and the anal valves segment XVIII (Fig. 9). Apart from a shift in segmental numbering, most of the Klompen et al. model for the posterior part of the opisthosoma would hold quite well. However, the model breaks down for the anterior idiosoma. Those authors did not consider the presence of an ocular segment, which in larval Ixodidae most likely takes up most of the scutum. Second, the scutal border is unlikely to coincide with a ‘‘true’’ segmental border (again, similar to the assumed condition in Opilioacarida). Considering the proposed designations for the posterior idiosoma, and the requirements associated with the presence of an ocular segment, one is left accommodating (dorsal) expression of segments VIII/IX [fused pregenital ? genital as proposed by Klompen et al. (1996)], X, and XI and perhaps VI and VII (segments associated with legs III–IV). Klompen et al. (1996) recognized three segments (their V, VI, VII/VIII) plus part of segment IV between the eyes and the festoons-associated segments. A possible match is shown in Fig. 9. The posterior delimitation of the ocular segment in Fig. 9 is not indicated, given that there are no available markers for its median posterior border. Similarly, the question whether the podal segments are expressed mid-dorsally cannot be resolved with available data. A more challenging problem is posed by Mesostigmata. Once again starting with the hypothesis that Mesostigmata share the presence of 18 primary segments, the following match between the standard nomenclature of Mesostigmata and the opilioacarid model is proposed (Fig. 10). The anal valves once again represent segment XVIII. Within Opilioacarida all immatures examined (4 genera) show an unpaired seta posterior to the anus, and two setae in the Zv position ventral to the anus. This pattern is remarkably similar to the positions of the postanal and paranal setae in many Mesostigmata. We therefore expand on an earlier suggestion by Lindquist (1994) that setae po and pa represent the pre-anal segment (XVII). Moving anterior, setal rows J1–J5 would correspond to segments XII– XVI. For lack of better terminology, J1 etc. in this context indicates the entire transverse series of J1, Z1, S1 and R1 setae (plus their ventral counterparts if present). Up to this point proposed homologies are relatively straightforward, after this they become more speculative. The setae corresponding to the podosomal j4–j6 rows may represent the dorsal elements of segments VIII–XI, with the j4 series forming the combined dorsal expression area of the pregenital and genital segments (VIII/IX). In this model, the area occupied by

123

Exp Appl Acarol

I

a

b

j1-3 (II-VII) j4

j5 VIII/IX j6 X J1

g

XI J2

XII

s ly

J3

XIII

J4 J5

XIV XV

XVI

XIV

pa XV

po XVI

XVII

Fig. 10 Mesostigmata: Lasioseius alli Chant, deutonymph, idiosoma. a Dorsum, b venter. Putative segmental homology—indicated by roman numbers; proposed segmental borders indicated by dashed lines. g gland, ly lyrifissure, pa paranal setae, po postanal seta, s seta. Redrawn and modified from Lindquist and Evans (1965)

setae of the j1–j3 series would be part of a reduced ‘‘ocular segment’’, and perhaps remnants of the ‘‘true’’ podal segments. The arrangement of especially the j4 and j5 series, with lateral members (s and r setae) usually inserted distinctly more posterior than the dorsal ones would be predicted under this hypothesis, reflecting the dorsal anterior ‘‘extension’’ of the opisthosomal segments. We stress that the above does not prove that the opilioacarid model of 18 segments is correct for all Parasitiformes, only that this model is not inconsistent with patterns observed in various parasitiform lineages, requiring only modest (and anticipated) segmental distortion to ‘‘fit’’.

Instar homology across Parasitiformes The current study also allows some comments on existing hypotheses on instar homology among the parasitiform lineages. The various lineages of Parasitiformes show quite distinctive post-embryonic developmental patterns. Opilioacarida retain the presumed ancestral sequence (also seen in basal Acariformes) of a prelarva, larva, three nymphs and the adult (Lindquist 1984). Holothyrida retain the three nymphal instars, but appear to lack a prelarva. Ixodid ticks have reduced this sequence further to a larva, one nymph and the adult, while Mesostigmata retain a larva, two nymphal instars and the adult. The condition in argasid ticks is difficult to interpret, as this group shows variable numbers of nymphs, depending on species, sex and nutritional status (Pound et al. 1986). Interestingly, many species do appear to end up with about three nymphal instars, the presumed ancestral number. One question is what instar(s) is/are missing in the various taxa with curtailed

123

Exp Appl Acarol

development. Loss of the prelarva in Holothyrida, Ixodida and Mesostigmata seems likely, all other hypotheses are less obvious. The current study does bring up an intriguing possibility: could the larva in Mesostigmata be serially homologous to the protonymph in Opilioacarida? In other words, could the missing instar in Mesostigmata be the larva rather than the tritonymph or adult as generally (e.g. Walter and Proctor 2013) assumed? This idea follows Sitnikova (1978), who suggested that reduction of early instars is a common phenomenon in Arachnida. The idea also appears to have been adopted by Andre´ and N’Dri (2013 (2012)). Some new observations are consistent with this hypothesis. The transition from ‘‘larva’’ to ‘‘protonymph’’ in Mesostigmata is characterized by the addition of a single seta on the palp trochanter and first appearance of R and Rv setae, changes shown to occur in Opilioacarida at the molt from proto- to deuto-nymph. Moreover, ‘‘larvae’’ in many Mesostigmata feed, while opilioacarid and (most) holothyrid larvae do not appear to do so (and have the regressed mouthparts to show it). An exception is made up by allothyrid larvae who have well developed chelicera and may feed. It would seem unusual that opilioacarid larvae lost that ability, while many mesostigmatid ones retained it (or gained it). Other evidence does not support this idea. The addition sequence of the circumbuccal and sternal setae, with st5 added on the genital capsules in the protonymph (as in Mesostigmata), as well as the addition sequence of stigmata (none in the larva, 1–2 pairs in the protonymph) is more consistent with the classical hypothesis. The ‘‘mesostigmatid larvae are homologous with opilioacarid protonymphs’’ hypothesis would also require the regression of legs IV in the mesostigmatid protonymph (the ‘‘larva’’ of current use), which is a substantial change, even if the level of regression of legs IV is clearly variable among taxa. For example, opilioacarid and holothyrid larvae express legs IV partially. Convincing evidence on this issue will probably have to come from embryology, specifically in the form of the presence of extra cuticles, representing ‘‘missing’’ instars. We are not aware of such evidence for Mesostigmata. A note in Aeschlimann and Hess (1984) listing 1–2 extra cuticles in ticks is intriguing, but needs confirmation. Acknowledgments For permission to examine specimens they collected, or in their collection, we thank Luis F. de Armas, Brian Fisher, Charles Griswold, Mark Harvey, Dania Prieto, Will Reeves, Owen Seeman, and Darryll Ubick. This work was supported in part by NSF Grant DEB-REVSYS 1026146 (HK), CUMEX (Consorcio de Universidades Mexicanas (MMV), and Capes/Brazil PDSE 1202-12-0 (LFOB).

References Aeschlimann A, Hess E (1984) What is our current knowledge of acarine embryology? In: Griffiths DA, Bowman CE (eds) Acarology VI, vol 1. Ellis Horwood Limited, Chichester, pp 90–99 Alberch P, Gould SJ, Oster GF, Wake DB (1979) Size and shape in ontogeny and phylogeny. Paleobiology 5:296–317 Alberti G (1980) Zur Feinstruktur der Spermien und Spermiocytogenese der Milben (Acari). II. Actinotrichida. Zool Jahrb Anat 104:144–203 Alberti G (2005) On some fundamental characteristics in acarine morphology. Atti Accad Naz Ital Entomol 53:315–360 Alberti G, Coons LB (1999) Volume 8C. Acari: Mites. In: Harrison FW, Foelix RF (eds) Microscopic anatomy of invertebrates. Chelicerate Arthropoda. Wiley, New York, pp 515–1215 Altner A, Prillinger L (1980) Ultrastructure of invertebrate chemo-, thermo-, and hygroreceptors and its functional significance. Int J Cytol 67:69–139 Andre´ HM, N’Dri JK (2013 (2012)) Bre´viaire de taxonomie des acariens, vol 13. Bruxelles, Belgique, Abc Taxa

123

Exp Appl Acarol Athias-Henriot C (1957) Phytoseiidae et Aceosejiidae (Acarina, Gamasina) d’Alge´rie. I. Genres Blattisocius keegan, Iphiseius Berlese, Amblyseius Berlese, Phytoseius Ribago, Phytoseiulus Evans. Bull Soc Hist Nat Afr Nord 48:319–352 Athias-Henriot C (1971) Un progre`s dans la connaissance de la composition me´tame´rique des gamasides: leur sigillotaxie idiosomale (Arachnida). Bull Soc Zool Fr 96:73–85 Bernardi LFdO, Klompen H, Ferreira RL (2013a) Adult growth in Opilioacaridae with 1904 (Acari: Parasitiformes: Opilioacarida). Ann Entomol Soc Am 106:788–790. doi:10.1603/AN13056 Bernardi LFdO, Klompen H, Zacarias MS, Ferreira RL (2013b) A new species of Neocarus Chamberlin & Mulaik, 1942 (Opilioacarida: Opilioacaridae) from Brazil, with remarks on postembryonic development. Zookeys 358:69–89. doi:10.3897/zookeys.358.6384 Bernardi LFdO, Silva FAB, Zacarias MS, Klompen H, Ferreira RL (2013c) Phylogenetic and biogeographic analysis of the genus Caribeacarus (Acari: Opilioacarida), with description of a new South American species. Invertebr Syst 27:294–306. doi:10.1071/IS12041 Camin JH, Clark GM, Gorirossi-Bourdeau F (1958) The palpal ‘‘tined seta’’ in the Mesostigmata, a homologue of the palpal claw in the Onychopalpida (Acarina).In: Proceedings of the tenth international congress of entomology, pp 903–908 Chamberlin RV, Mulaik S (1942) On a new family of Notostigmata. Proc Biol Soc Wash 55:125–132 Coineau Y (1973) A propos de quelques caracte`res particulie`rement primitifs de la pre´larva et de la larva d’un Opilioacaridae du Gabon (Acariens). C R Acad Sci Paris 276:1181–1184 Coineau Y, Legendre R (1975) Sur un mode de re´ge´ne´ration appendiculaire ine´dit chez les Arthropodes: la re´ge´ne´ration des pattes marcheuses chez les Opilioacariens (Acari: Notostigmata). C R Acad Sci Paris 280:41–43 Coineau Y, Van der Hammen L (1979) The postembryonic development of Opilioacarida, with notes on new taxa and on a general model for the evolution. In: Piffl E (ed) Proceedings 4th international congress of acarology, Saalfelden (Austria), Akade´miai Kiado´, Budapest, pp 437–441 Damen WGM, Hausdorf M, Seyfarth E-A, Tautz D (1998) A conserved mode of head segmentation in arthropods revealed by the expression pattern of Hox genes in a spider. Proc Natl Acad Sci USA 95:10665–10670 Dunlop JA, Alberti G (2007) The affinities of mites and ticks: a review. J Zool Syst Evol Res. doi:10.1111/j. 1439-0469.2007.00429.x Evans GO (1992) Principles of acarology, 1st edn. CAB international, Wallingford Grandjean F (1936) Un acarien synthe´tique: Opilioacarus segmentatus With. Bull Soc Hist Nat Afr Nord 27:413–444 Grandjean F (1954) E´tude sur les Palaeacaroides (Acariens, Oribates). Me´m Mus nat Hist natur (NS), se´r A Zool 7:179–272 Grandjean F (1969) Stases. Actinopiline. Rappel de ma classification des acariens en 3 groupes majeurs. Terminologie en soma. Acarologia 11:796–827 Johnston DE (1982) Opilioacariformes, Parasitiformes. In: Parker SB (ed) Synopsis and classification of living organisms. McGraw-Hill, New York, pp 111–117 Klompen H (1992) Comparative morphology of argasid larvae (Acari: Ixodida: Argasidae), with notes on phylogenetic relationships. Ann Entomol Soc Am 85:541–560 Klompen H (2000) Prelarva and larva of Opilioacarus (Neocarus) texanus (Chamberlin & Mulaik) (Acari: Opilioacarida) with notes on the patterns of setae and lyrifissures. J Nat Hist 34:1977–1992. doi:10. 1080/00222930050144819 Klompen H (2010) Holothyrids and ticks: new insights from larval morphology and DNA sequencing, with the description of a new species of Diplothyrus (Parasitiformes: Neothyridae). Acarologia 50:269–285. doi:10.1051/acarologia/20101970 Klompen H, Keirans JE, Filippova NA, Oliver JH Jr (1996) Idiosomal lyrifissures, setae, and small glands as taxonomic characters and potential indicators of ancestral segmentation patterns in larval Ixodidae (Acari: Ixodida). Int J Acarol 22:113–134 Klompen H, Lekveishvili MG, Black WC IV (2007) Phylogeny of parasitiform mites (Acari) based on rRNA. Mol Phylogenet Evol 43:936–951. doi:10.1016/j.ympev.2006.10.024 Krantz GW (2009) Form and Function. In: Krantz GW, Walter DE (eds) A manual of acarology, 3rd edn. Texas Tech University Press, Lubbock, pp 5–53 Leclerc P (1989) Considerations pale´oge´ographique a` propos de la de´couverte en Thaı¨lande d’Opilioacariens nouveaux (Acari-Notostigmata). C R Soc Bioge´ogr 65:162–174 Lindquist EE (1984) Current theories on the evolution of major groups of Acari and on their relationship with other groups of Arachnida, with consequent implications for their classification. In: Griffiths DA, Bowman CE (eds) Acarology VI, vol 1. Ellis Horwood Limited, Chichester, pp 28–62

123

Exp Appl Acarol Lindquist EE (1994) Some observations on the chaetotaxy of the caudal body region of gamasine mites (Acari: Mesostigmata), with a modified notation for some ventrolateral body setae. Acarologia 35:323–326 Lindquist EE, Evans GO (1965) Taxonomic concepts in the Ascidae, with a modified setal nomenclature for the idiosoma of the Gamasina (Acarina: Mesostigmata). Mem Entomol Soc Can 47:1–64 Lindquist EE, Krantz GW, Walter DE (2009) Classification. In: Krantz GW, Walter DE (eds) A manual of acarology, 3rd edn. Texas Tech University Press, Lubbock, pp 97–103 Moraza ML (2005) Tarsus I chaetotaxy and structure in Anactinotrichida mites: characters with phylogenetic value. In: Weigmann G, Alberti G, Wohltmann A, Ragusa S (eds) Acarine biodiversity in the natural and human sphere, vol XIV. Phytophaga, Palermo, pp 347–359 Murrell A, Dobson SJ, Walter DE, Campbell NJH, Shao R, Barker SC (2005) Relationships among the three major lineages of the Acari (Arthropoda : Arachnida) inferred from small subunit rRNA: paraphyly of the Parasitiformes with respect to the Opilioacariformes and relative rates of nucleotide substitution. Invertebr Syst 19:383–389 Naudo MH (1963) Acariens Notostigmata de l’Angola. Publ Cult Co Diam Ang, Lisboa 63:13–24 Norton RA, Kethley JB, Johnston DE, OConnor BM (1993) Phylogenetic perspectives on genetic systems and reproductive modes of mites. In: Wrensch DL, Ebbert MA (eds) Evolution and diversity of sex ratio in insects and mites. Chapman & Hall, New York, pp 8–99 Pepato AR, da Rocha CEF, Dunlop JA (2010) Phylogenetic position of the acariform mites: sensitivity to homology assessment under total evidence. BMC Evol Biol. doi:10.1186/1471-2148-10-235 Pound JM, Campbell JD, Andrews RH, Oliver JH Jr (1986) The relationship between weights of nymphal stages and subsequent development of Ornithodoros parkeri (Acari: Argasidae). J Med Entomol 23:320–325 Santos VT, Ribeiro L, Fraga A, de Barros CM, Campos E, Moraes J, Fontenele MR, Araujo HM, Feitosa NM, Logullo C, Nunes da Fonseca R (2013) The embryogenesis of the tick Rhipicephalus (Boophilus) microplus: the establishment of a new chelicerate model system. Genesis 51:803–818. doi:10.1002/ dvg.22717 Scholtz G, Edgecombe GD (2006) The evolution of arthropod heads: reconciling morphological, developmental and palaeontological evidence. Dev Genes Evol 216:395–415. doi:10.1007/s00427-0060085-4 ¨ ber die Ko¨rpergliederung der Zecken, die Zusammenzetzung des Gnathosoma und die Schulze P (1932) U Beziehungen der Ixodoidea zu den fossilen Anthracomarti. Sitz ber Abh Naturf Ges Rostock 3:104–126 Shultz JW (1989) Morphology of locomotor appendages in Arachnida: evolutionary trends and phylogenetic implications. Zool J Linn Soc 97:1–56 Shultz JW (2007) A phylogenetic analysis of the arachnid orders based on morphological characters. Zool J Linn Soc 150:221–265 Sitnikova LG (1978) The main evolutionary trends of the Acari and the problem of their monophyletism. Entomol Obozr 57:431–457 Telford MJ, Thomas RH (1998) Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment. Proc Natl Acad Sci USA 95:10671–10675 Van der Hammen L (1966) Studies on Opilioacarida (Arachnida). I. Description of Opilioacarus texanus (Chamberlin & Mulaik) and revised classification of the genera. Zool Verhand, Leiden 86:3–80 Van der Hammen L (1968) Studies on Opilioacarida (Arachnida). II. Redescription of Paracarus hexophthalmus (Redikorzev). Zool Meded, Leiden 43:57–76 Van der Hammen L (1969) Studies on Opilioacarida (Arachnida). III. Opilioacarus platensis Silvestri, and Adenacarus arabicus (With). Zool Meded, Leiden 44:113–131 Van der Hammen L (1970) La segmentation primitive des Acariens. Acarologia 12:3–10 Van der Hammen L (1977) Studies on Opilioacarida (Arachnidea). IV. The genera Panchaetes Naudo and Salfacarus gen. nov. Zool Meded, Leiden 51:43–78 Van der Hammen L (1989) An introduction to comparative arachnology. SPB Academic Publishing, The Hague Va´zquez MM, Klompen H (2002) The family Opilioacaridae (Acari: Parasitiformes) in North and Central America, with description of four new species. Acarologia 42:299–322 Va´zquez MM, Klompen H (2009) New species of New World Opilioacaridae (Acari: Parasitiformes) with the description of a new genus from the Caribbean region. Zootaxa 2061:23–44 Va´zquez MM, Klompen H (2010) The genus Salfacarus (Acari: Opilioacarida) in Madagascar. Zootaxa 2482:1–21 Va´zquez MM, Palacios-Vargas JG (1988) Algunas observationes sobre el comportamiento de los acaros opilioacaridos (Acarida: Notostigmata). Rev Nica Ent 6:1–6

123

Exp Appl Acarol Walter DE, Proctor HC (1998) Feeding behaviour and phylogeny: observations on early derivative Acari. Exp Appl Acarol 22:39–50. doi:10.1023/A:1006033407957 Walter DE, Proctor HC (1999) Mites: ecology, evolution and behaviour. CABI Publishing, New York Walter DE, Proctor HC (2013) Mites: ecology, evolution & behaviour, 2nd edn. Springer, Dordrecht Weigmann G (2001) The body segmentation of oribatid mites from a phylogenetic perspective. In: Halliday RB, Walter DE, Proctor HC, Norton RA, Colloff MJ (eds) Acarology: proceedings of the 10th international congress, CSIRO Publishing, Melbourne, pp 43–49 Wolff C, Hilbrant M (2011) The embryonic development of the central American wandering spider Cupiennius salei. Front Zool 8:1–34

123