Shedding light on a cryptic cavernicole: A second species of Zenkevitchia Birstein (Crustacea, Amphipoda, Typhlogammaridae) discovered via molecular techniques Author Sidorov, Dmitry A. Author Gontcharov, Andrey A. Author Palatov, Dmitry M. Author Taylor, Steven J. Author Semenchenko, Alexander A. text Subterranean Biology 2015 15 37 55 http://dx.doi.org/10.3897/subtbiol.15.4872 journal article http://dx.doi.org/10.3897/subtbiol.15.4872 1314-2615-15-37 1C55AB5B2AA2413B9F101B7DA2F87F8D Taxon classification Animalia Amphipoda Typhlogammaridae Zenkevitchia yakovi Sidorov sp. n. Figures 2, 3, 4, 5, 6, 7 Diagnosis (both sexes). Robust, small-sized species of gammarid-like habitus (sexual dimorphism marked, i.e., some males larger than females, gnathopods 1-2 and uropod 3 sexually dimorphic). Antenna 1 short, reaching 50% length of body; antenna 2 short, reaching about 40-60% length of antenna 1, gland cone long, reaching half of peduncle segment 4. Maxilla 1 inner plate triangular with 20-24 plumose setae, outer plate of filtration-type, with 105-114 multi-toothed sickle-shaped comb-like spines; palps symmetrical and reduced. Maxilla 2 inner and outer plates apically with numerous setae in two rows. Mandibular palp article 3 with 1 A group of 2 setae, 2 B setae, 13 D setae, 4-5 E setae. Gnathopods 1-2 small, with propodus not larger than corresponding coxa; palmar angles of both gnathopods with singly notched, strong corner spine on inner face; dactyli with 1 seta along outer margin, 1 stiff seta at nail base along inner margin, nails short; inner margin of dactyli of pereopods 3-7 with 1 stiff seta, with 1 thin seta at nail base. Urosomites 1-2 with dorsolateral groups of spines. Pleopods 1-3 with 2 coupling setae (retinacula) each accompanied with 1 strong, serrate spine (2+1). Uropod 1 rami almost equal, inner ramus scarcely shorter than outer ramus. Uropod 3 short, as long as uropod 2, endopodite small, 0.4 -0.5x shorter than exopodite. Telson with 1 or 2 distal spines per lobe. Coxal gills 2-7 stalked, triangular or sacciforme, largest on gnathopod 2, successively smaller on pereiopods 3 to 7, gill 7 the smallest. Body length 8.0-9.75 mm (females), 5.0-10.0 (males). Type material. Holotype: male, 10.0 mm, X43382/Cr-1613-FEFU, completely dissected and mounted on a single glass slide. Abkhazia, Gulripshi district, Tsabal, "Istocnik Tcebel'da" Cave, 43.026216, 41.283022, cave pool, 31.01.2012, coll. D.M. Palatov. Paratypes: X43383/Cr-1614-FEFU, 1 male, 8.0 mm, 1 female, 8.5 mm (oostegites weakly differentiated), with same data as holotype. Additional material examined (not placed in the type series). All specimens measured, partially dissected and stored in vial (1-11/1sd-IBSS), 4 females with oostegites not well differentiated (2x8.0 mm, 8.5 mm, 9.75 mm), 9 males (3x8.0 mm, 4x8.5 mm, 9.75 mm, 5.0 mm), 1 juvenile (3.0 mm); with same data as holotype; ~ 4.1 km E of "Istocnik Tcebel'da" Cave, Sredne-Shakuranskaya Cave, 43.029748, 41.333198, small cave river, 30.01.2012, coll. D.M. Palatov. Etymology. The specific epithet yakovi (Latin) refers to the first name of Yakov Avadievich Birstein a famous zoologist is known for his outstanding contributions to the systematics of subterranean crustaceans. Description. Male, 10.0 mm (X43382/Cr-1613-FEFU). General body morphology (Figures 2, 3H, 4A, H, 7E). Body smooth, lacking dorsal cuticular elements (keel or tubercles) on pereon and pleon. Head as long as first pereon segment; lack rostrum; inferior antennal sinus shallow, sub-rounded. Eyes absent. Pereonite 7, pleonites 1-3 and uronite 1 with median and lateral groups of thin setae. Urosomites 1 and 2 on dorsal surface with lateral groups of spines accompanied with setae. Epimeral plate 1: postero-ventral corner acuminate; posterior and ventral margins convex; 1 stiff setae along ventral margin, 3 setae along posterior margin. Epimeral plate 2: postero-ventral corner acuminate; posterior margin straight; ventral margin convex; 3 notched spines along ventral margin, 4 setae along posterior margin. Epimeral plate 3: postero-ventral corner acuminate; posterior margin slightly concave; ventral margin convex; 4 notched spines along ventral margin, 3 setae along posterior margin. Telson width: length ratio is 1: 0.85; cleft 0.9 of length; only 2 sub-apical spines per lobe present, these are 0.25 -0.30x telson length, each accompanied by 1 or 2 plumose seta. Antennae (Figures 2, 4B, C). Antenna 1 0.5 x of body length; flagellum with up to 27 articles; each article with 5-6 short setae; peduncular articles in ratio 1: 0.8: 0.5; proximal article of peduncle distally with 2 medial sets of long setae; accessory flagellum 4-articulated. Length ratio antenna 1: 2 as 1: 0.57; flagellum of antenna 2 with 11 articles, each article densely setose; peduncle articles lengths 4: 5 is 1: 0.8; flagellum slightly longer than peduncle (articles 4+5); peduncular articles 4 and 5 with sets of long stiff setae laterally; gland cone long, reaching half of peduncle segment 4. Mouth parts, typical gammarid except for unusual filtration-type maxilla 1 (Figures 4 D-G , 5 A-H ). Labrum rounded, clypeus detached, longer than broad. Inner lobes of labium absent, outer lobes broad with stiff curved setae marginally, mandibular process distinct (narrow). Left mandible: incisor with 5 teeth, lacinia mobilis with 4 teeth; between lacinia and molar row of 7 serrate spines. Right mandible: incisor process with 4 teeth, lacinia mobilis bifurcate with several small denticles, between lacinia and molar a row of 8 serrate spines; triturative molar process with long lanose seta. Mandibular palp article 2 slightly longer than article 3 (distal); proximal palp article without setae; the second article with 13 setae; distal article narrowed, with 1 A group of 2 setae, 2 B setae, 13 D setae, 4-5 E setae. Maxilla 1 palp reduced, distal article with 5 or 6 apical strong serrate setae (both palps symmetrical); outer plate with 114 multi-toothed sickle-shaped spines; inner plate triangular with 24 plumose setae. Maxilla 2 inner plate smaller than outer one with oblique row of 24 plumose setae; both of them apically with numerous setae in two rows. Maxilliped palp article 2 with about 70 setae along inner margin; article 4 (distal) with dorsal seta, bearing 5 seta at the nail base, nail shorter than pedestal; outer plate with 15 flattened spines and 9 long plumose setae on apex; inner plate with 3 strong spines and 5 stiff naked setae on apex, 26 plumose setae on ventral face, 23 stiff denticulate setae in 3 rows on dorsal face. Coxal plates, gills (Figures 3A, C, 6A, C, E, G, I). Coxal plate 1 of rectangular shape, antero-ventral margin extended with 3 setae. Coxal plate 2 of rectangular shape, antero-ventral margin narrowed with 5 setae. Coxal plate 3 width: depth is 0.5: 1; along antero-ventral margin 4 setae. Coxal plate 4 of sub-quadrate shape, width: depth is 0.9: 1; posteriorly with prominent excavation; along ventral margin 10 setae. Coxal plates 5-7 progressively smaller towards the posterior; coxal plates 5-6: only anterior lobe well-developed; posterior margin simple or pointed with 1 seta. Coxal plate 7 semicircular, along posterior margin 3 setae in shallow serration. Coxal gills 2-7 stalked, large but progressively smaller towards the posterior; gills 2-4 triangular, gills 5-7 saccular or irregularly ovoid. Gnathopods 1 and 2 (Figures 3 A-D ). Gnathopod 1, ischium with postero-distal set of setae. Carpus 0.4 x length of basis and 0.57 x length of propodus; anterior margin of carpus with two group of setae; carpus posteriorly with transverse row of lateral and sub-marginal setae. Propodus pyriform, palm straight with cutting margin acanthaceous and shorter than posterior margin; along posterior margin 3 sets of simple setae; anterior margin with 3 pairs of setae, antero-distal group with 7 setae; palmar margin with short, notched setae along outer and inner faces and armed with row of 4 distally notched, robust spines on inside and 5 on outside, palmar angle with 1 strong corner spine on inner face; nail 0.3 x total length of dactylus, a 1 seta along anterior margin, 1 short stiff seta along inner margin and 3 setules at hinge. Gnathopod 2, basis width: length is 0.29: 1. Ischium with 4 sets of postero-distal setae. Carpus 0.35 x length of basis and 0.5 x length of propodus; anterior margin of carpus with 1 distal set of setae; carpus posteriorly with 5 lateral sets of setae. Propodus small (compared to the body) and 1.14 x larger than propodus of gnathopod 1; propodus sub-quadrate, palm straight with cutting margin acanthaceous and shorter than posterior margin; posterior margin with 7 rows of simple setae; anterior margin with 2 sets of setae; antero-distal group with 8 setae; palmar margin with short, notched setae along outer and inner faces and armed with row of 4 distally-notched robust spines on inside and 2 on outside, palmar angle with 1 strong corner spine on inner face; dactylus similar to that of gnathopod 1. Pereopods 3-7 (Figures 6 A-J ). Lengths of pereopods 3-4 equal. Dactylus 4 0.4 x propodus 4; nail length 0.4 x total dactylus length. Dactyli 3-4 with dorsal plumose seta; inner margin with 1 stiff seta and 1 thin seta at hinge. Lengths of pereopods 5: 6: 7 is 1: 1.1: 0.9. Pereopod 7 length 0.45 x body length. Bases 5-7 narrowed distally, length: width is 1: 0.77-0.92; posterior margin convex (distinctly in basis 7), without postero-distal lobes; posteriorly marginal serrations with long setae (expressed in basis 5); anteriorly 5-7 notched spines. Dactylus 7 length 0.3 x propodus 7 length. Dactyli 5-7 with dorsal plumose seta; inner margin with 1 stiff seta and 1 thin seta at hinge. Pleopods and uropods (Figures 2, 3 E-G , 4I, J). Pleopods 1-3 sub-equal, each with 2 coupling setae accompanied by 1 strong, serrate spine; peduncular articles fringed with long, thin setae; proximal article of inner rami fringed with 3-4 bifurcate setae. Pleopods 1-3 rami with 12-15 articles each. Uropod 1 protopodite with 1 basofacial spine, 4 dorso-lateral spines and 1 dorso-medial spine; exopodite: endopodite length is 1: 0.9; rami straight with single spines along outer margins; both with 5 strong notched spines apically and sub-apically. Uropod 2 exopodite as long as endopodite. Uropod 3 protopodite with two groups of strong notched spines on apex; endopodite 0.6 of protopodite length, with 2 spines and 3-5 long setae apically; exopodite 1.5 x longer than protopodite, with 3 groups of lateral spines, long simple setae along inner margin, 5 spines and 10 long setae apically. Figure 2. Photograph of live specimen of Zenkevitchia yakovi sp. n. in the cave "Istocnik Tcebel'da" , from right side. Photography by A. Korotaev. Figure 3. Zenkevitchia yakovi sp. n.: holotype, male (10.0 mm), X43382/Cr-1613-FEFU. A Gnathopod 1, lateral view B Enlarged palmar part of gnathopod 1 C Gnathopod 2, lateral view D Enlarged palmar part of gnathopod 2 E, F, G Uropods 1-3, dorsal views H Telson, dorsal view. Figure 4. Zenkevitchia yakovi sp. n.: holotype, male (10.0 mm), X43382/Cr-1613-FEFU. A Head, lateral view B, C Antenna 1 and 2, lateral views D Labium, ventral view E Labrum, anterior view F, G Left and right mandibles, medial views H Epimeral plates 1-3, lateral views I pleopod 2, medial view J Coupling setae (retinacula), medial view. Figure 5. Zenkevitchia yakovi sp. n.: holotype, male (10.0 mm), X43382/Cr-1613-FEFU. A Maxilliped, ventral view B, C Enlarged outer and inner plates of maxilliped, ventral view D Enlarged inner plate of maxilliped, dorsal view E Left maxilla 1, dorsal view F Enlarged outer plate of maxilla 1 G Palp of right maxilla 1, dorsal view H Maxilla 2, dorsal view. Figure 6. Zenkevitchia yakovi sp. n.: holotype, male (10.0 mm), X43382/Cr-1613-FEFU. A, C, E, G, I Pereopods 3-7, lateral views B, D, F, H, J Enlarged dactyli of pereopods 3-7, lateral views. Figure 7. Zenkevitchia yakovi sp. n.: paratype, female (8.5 mm), X43383/Cr-1614-FEFU. A Gnathopod 1, medial view B Enlarged palmar part of gnathopod 1 C Gnathopod 2, medial view D Enlarged palmar part of gnathopod 2 E Metasome and urosome, lateral view F, G Uropods 1 and 2, dorsal views H Uropod 3, ventral view I Telson, dorsal view. Female, 8.5 mm (X43383/Cr-1614-FEFU), sexually dimorphic characters (Figures 7 A-I ). Body smaller than male, with more slender body. Antenna 1 50% of total body length; antenna 2 short reaching 40% length of antenna 1. Gnathopods 1 and 2 propodi similar, sub-rectangular, palmar margins sub-transverse, short and weakly armed with spines. Carpus of gnathopod 2 flexible, as long as propodus. Uropod 3 protopodite lacking lateral group of apical spines; exopodite 2 x longer than protopodite. Oostegites 2-5 on gnathopod 2 and pereopods 3-5. Variability. The pilosity of posterior margin of pereopod 5 bases is very variable, ranging from normally setose to very densely setose. The form of postero-ventral corner and number of spines on ventral margin of epimeral plates varies slightly. Maxilla 1 distal palp article with 5-8 apical setae; outer plate with 105-114 spines; inner plate with 20-24 setae. Endopodite of uropod 3 with 1 or 2 spines on apex. The propodus of gnathopod 2 on female specimens from the Sredne-Shakuranskaya Cave were somewhat elongated, with palmar margin distinctly transverse; rami of uropod 2 of both sexes unarmed along outer margins. One specimen was observed in sample from the "Istocnik Tcebel'da" Cave with the same character of uropod 2 rami. Remarks. Taxonomy of the genus Zenkevitchia is confusing and requires a thorough revision. The first mention of Zenkevitchia admirabilis was given by Birstein in 1940 , who gave a description and drawings of a male (14.0 mm body length), but without specifying the geographical origin of the material. However, a year later Birstein (1941) provided a detailed description of the genus as well as the species Zenkevitchia admirabilis , based on a 4.0 mm female from a cave near Andreevka (Apra), not far from Sukhumi. Further, Birstein and Ljovuschkin (1970) provided an expanded description of Zenkevitchia admirabilis and provided information on its distribution within Abkhazia. A detailed analysis of these species descriptions has left the impression that Zenkevitchia admirabilis is represented by a series of different species in this region. Indirectly, this is evidenced in remarks by Birstein and Ljovuschkin (1970 , p. 1473, [here translated from Russian]): "The study of a large specimens (up to 20 mm in length) suggests a significant age-related variation in this species, description of which is done on the basis of a female of 4 mm length and supplemented by a 14 mm male. " ..." with increases in body size, there are changes in the armament of mouthparts, gnathopods and probably the number of spines on urosomal segments". Due to the fact that the 14.0 mm male ( Birstein 1940 ) and 4.0 mm female ( Birstein 1941 ) belongs to a different species (Table 1) and both descriptions are very poor we consider that detailed comparison is premature in this work. The searching of type series from cave near Andreevka was not successful, and the cave was mined in 1992-93 during the Georgian-Abkhaz conflict (Turbanov, pers. comm., 2015), therefore, a complete revision of the Zenkevitchia admirabilis species-complex is not yet feasible. Comparison of Zenkevitchia yakovi sp. n. with available descriptions of Zenkevitchia admirabilis did not allow us to identify any significant morphological differences, except for probably unimportant differences in body size and questionable dispersion in outer plate of maxilla 1, bearing ≥ 23-50 spines for Zenkevitchia admirabilis (see Table 1). We cannot excluded the possibility that the relatively small number of spines on the outer plate of maxilla 1 in the original descriptions may be the result of Birstein not using a crushing technique to reveal additional spines. Based on the fact that Zenkevitchia yakovi sp. n. fits within the ranges of morphological variability indicated by Birstein for Zenkevitchia admirabilis ( Birstein and Ljovuschkin 1970 ; Table 1), but genetically both lineages are quite distinct, we feel the hidden diversity within Zenkevitchia requires examination of additional material from other localities. Table 1. Variation in morphological characters in adult specimens of Zenkevitchia yakovi sp. n. and comparison with published data for Zenkevitchia admirabilis .

Character	Zenkevitchia yakovi sp. n.	Zenkevitchia admirabilis Birstein, 1940	Zenkevitchia admirabilis (Birstein, 1941)	Zenkevitchia admirabilis (Birstein & Ljovuschkin, 1970)
1

Table 2. List of the specimens, sampling sites and accession numbers of the sequences for COI included in this study. References are given for sequences obtained from GenBank.

Specimen	Locality	GenBank acc. no.	Reference / or year of sampling
Zenkevitchia yakovi	43.026216 N 41.283022 E	KP844572
Zenkevitchia yakovi	43.029748 N 41.333198 E	KP844573
Zenkevitchia yakovi	43.029748 N 41.333198 E	KP844574
Accubogammarus	KF478592	Hou et al. (2014)
Anopogammarus revazi	KF478522	Hou et al. (2014)
Metohia carinata	KF478584	Hou et al. (2014)
Typhlogammarus mrazeki	KF478590	Hou et al. (2014)
Typhlogammarus	KF478591	Hou et al. (2014)
Zenkevitchia admirabilis	KF478600	Hou et al. (2014)
Zenkevitchia admirabilis	KF478599	Hou et al. (2014)

Table 3. Estimates of pairwise sequence divergence (uncorrected p-distances) of partial mitochondrial COI gene among species and haplotypes of the Balkan and Caucasian Typhlogammaridae .

Zenkevitchia yakovi

Zenkevitchia yakovi

Zenkevitchia yakovi

Zenkevitchia admirabilis

Zenkevitchia admirabilis

Accubogammarus

Typhlogammarus

Typhlogammarus mrazeki

Metohia carinata

Anopogammarus revazi