Paradoxiclamousella pirata, Camacho & Dorda & Rey, 2013

Camacho, A. I., Dorda, B. A. & Rey, I., 2013, Old and new taxonomic tools: description of a new genus and two new species of Bathynellidae from Spain with morphological and molecular characters, Journal of Natural History (J. Nat. Hist.) 47 (21 - 22), pp. 1393-1420 : 1408-1415

Paradoxiclamousella pirata gen. nov. sp. nov.

( Figures 4–6 )

Material examined

Type locality. Río Chico cave , Soba (Cantabria, Spain); coordinates 43.28383 N, 4.52318 W and Z: 575 ( WGS84 ); 4 June 2002 (six females and three males) nine specimens collected belonging to the type series, together with four DNA extractions from the specimens collected on 21 June 2011, which were used in molecular analysis (see voucher number on Table 4). Other localities: Becerral or Santos cave, Soba (Cantabria); coordinates 43.19491 N, 3.58453 W and Z: 685 ( WGS84 ); 4 June 2002 (one female) .

Details of the descriptions are based on all specimens collected in 2002. The holotype is a male ( MNCN 20.04 / 8877), the allotype is a female ( MNCN 20.04 / 8878) and the morphological type series contains two males and five females ( MNCN 20.04 / 8879 to 8885) .

Description

Body. Total length of holotype 0.75 mm and paratype 0.77 mm. Total length of male 0.70–0.85 mm, of female 0.71–0.86 mm. Body elongated, approximately nine times as long as wide, segments slightly widening towards posterior end. Rectangular head, longer than wide. Pleotelson with one very long barbed dorsal seta on each side. All drawings are of the holotype (male) except for female Th VIII, and one figure of Md that belong to the allotype.

Antennule ( Figure 4A ). Seven-segmented; length of first three segments similar to other four segments; fourth and fifth segments, similar and smaller than sixth; seventh segment longer than sixth; inner flagellum rectangular; setation as in Figure 1A ; segment six with two terminal aesthetascs and seventh with three aesthetascs; antennule slightly longer than the antenna.

Antenna ( Figure 4B ). Eight-segmented; slightly shorter than the antennule; the two terminal segments longer than the others and the last the longest; length of last four segments twice the length of the first four segments; second segment with small spines; ventromedial seta of exopod present and with two apical setae, one of these bifurcated sensory seta; setal formula: 0 / 0 / 2 + exp / 2 + 0 / 1 + 0 / 0 + 0 / 2 + 2 / 4.

Paragnath ( Figure 4C ). Lengthened and rounded in the distal part with thin apical setulation.

Mandible ( Figure 4D,E ). Palp with three segments, terminal segment with two equal and barbed claws; segment one small and almost square, segment two long and third very small. Masticatory part ( Figure 4E ): pars molaris with three main teeth, the two first simple (the nearest to processus incisivus accessorius with one tooth) the third tooth with small teeth on each side and incisor process (pars incisiva) with two teeth.

Maxillule ( Figure 4F ). Proximal endite with four setae; distal endite with six teeth, four with denticles and two more smooth, seta-like, and with three plumose setae on the outer margin, of different size.

Maxilla ( Figure 4G ). Four segments; setal formula 4, 4, 6, 6.

Thoracopods I–VII ( Figure 5A,G ). Th I ( Figure 5A ) slightly smaller than others; length gradually increasing from I to V; exopod similar in all thoracopods; Th VI ( Figure 5F ) to VII ( Figure 5G ) slightly longer than others but only the endopod, exopod just beyond the first two segments of endopod. Th I without epipod; coxa with a long and strong plumose seta; basipod with two long plumose setae and with a group of ctenidia. Basipod of Th II with two long plumose setae and with one smooth seta in Th III to VII. Exopod one-segmented on all thoracopods; with five barbed setae and with groups of ctenidia at base of setae. Endopod with three segments in Th I to V, and four segments in Th VI and VII; all setae are smooth except the setae on the distal outer corner of segment two that is plumose; with groups of ctenidia at lateral internal edge in all segments. Thoracopod endopod setal formulae: Th I, 3 + 0 / 4 + 1 / 4; Th II: 1 + 0 / 2 + 1 / 4; Th III: 1 + 0 / 2 + 1 / 4; Th IV: 1 + 0 / 2 + 1 / 4; Th V: 0 + 0 / 1 + 1 / 3; Th VI: 0 + 0 / 0 + 1 / 0 + 0 / 2(1); Th VII: 0 + 0 / 0 + 1 / 0 + 0 / 2(1).

Male thoracopod VIII ( Figure 6A,B). Elongated outer lobe (O. lb.), slightly shorter than the frontal projection; small outer protuberance (O. prt.); frontal projection (Fr. prj.) elongated, the longest of all the lobes of the penial region and does not reach the end of the basipod; inner lobe (I. lb.) rounded, shorter than the outer lobe; vertical trapezoid basipod (Bsp.) without setae; exopod with three terminal setae and a fourth medial seta; endopod not very small, half the size of the exopod, with two small terminal setae of similar length.

Female thoracopod VIII ( Figure 6C). Coxa with one smooth lateral seta; large epipod, similar to basipod; endopod smaller than exopod, with two smooth apical setae, one of these very long; exopod almost as basipod, two times longer than endopod, and with two apical smooth seta of different lengths.

First pleopod ( Figure 6D). Two-segmented, first segment with one long seta; second segment with five setae, all smooth.

Uropod ( Figure 6F). Sympod almost square, 1.3 times longer than endopod, with four strong equal spines; endopod almost twice as long as exopod, with two strong claws, the distal slightly longer, terminally with two barbed setae, one of these very long and with two barbed shorter setae located dorsolaterally; exopod with two terminal setae, the internal very long and another sub-basal seta.

Pleotelson ( Figure 6E). With one long, barbed dorsal seta on either side near base of furca.

Furcal rami ( Figure 6E). Almost square, bearing five spines; dorsal spine, similar to first and second spines, which are half length of the third spine, the longest, which is 1.5 times longer than fourth.

Etymology

The species name, pirata , substantive in apposition, to remember “the old pirate sailors”.

Remarks

The differences between P. pirata sp. nov. and P. fideli sp. nov. are subtle, but there are many. Species are similar in size, being relatively large for the family (around 0.5 mm other genera). Antennules and antenna have different size ratios between the segments, and different number and length of setae (see Figures 1A,B and 3A,B). The paragnaths have different patterns of setules. The mandibular palp is very strong in P. pirata sp. nov. and the terminal claws are barbed. The two setae of basipod of Th I and II are barbed in P. pirata sp. nov. and smooth in P. fideli sp. nov. The endopod of Th I has more setae in P. pirata sp. nov. than in P. fideli sp. nov. The third segment of endopod of Th II to V has only three setae in P. fideli sp. nov. and four on Th II to IV (three on Th V) in P. pirata sp. nov. The exopod of Th I to VII has four setae in P. fideli sp. nov. and five setae in P. pirata sp. nov. The relationship between size of the endopod and exopod of the thoracopods is different in both species (see Figures 2 and 4 ). The male Th VIII is larger in P. pirata sp. nov. than in P. fideli sp. nov.; outer protuberance and inner lobe are different in both species (see Figure 3A,B and Figure 6 A,B), whereas the outer lobe is similar in the two species. The basipod is rectangular and with a seta in P. fideli sp. nov. and almost square, without seta, in P. pirata sp. nov. and endopod is smaller in P. fideli sp. nov. than in P. pirata sp. nov. The epipod and coxal seta of the female Th VIII are longer in P. fideli sp. nov. than in P. pirata sp. nov.; the exopod and the endopod are longer in P. pirata sp. nov. than in P. fideli sp. nov. and endopod is very small in P. fideli sp. nov. and with a single seta (two setae in P. pirata sp. nov.). Pleopods are slightly different in both species and with six setae in P. pirata sp. nov. and five in P. fideli sp. nov. There is a different ratio between sympod and endopod of uropod in both species; sympod spines are longer and thicker in P. pirata sp. nov. than in P. fideli sp. nov. The dorsal seta of the pleotelson is twice as long in P. pirata sp. nov. as in P. fideli sp. nov. The furcal spines are of different sizes: the third is slightly longer than the others in P. fideli sp. nov. and the third is twice as long as the first and second in P. pirata sp. nov.

Molecular results

Nucleotide analysis

The alignment of all bathynellacean COI gene sequences (21 specimens in total) resulted in a consensus length of 508 bp. The models of sequence evolution selected for the mtDNA were GTR + I + G. No stop codons or gaps were observed in any of the translated amino acid sequences, suggesting that the genuine mtDNA COI gene was sequenced. On average, a bias towards A–T (69%) was observed in the bathynellacean sequences, which is consistent with previous results from parabathynellids and other crustaceans ( Guzik et al. 2008). The base frequencies were as follows: A = 0.28, C = 0.15, G = 0.16 and T = 0.40.

Genetic divergences

The uncorrected sequence divergence estimates between the specimens and the outgroup taxa are summarized in Table 1. The genetic divergence between the outgroup used, Iberobathynella burgalensis Camacho, 2005 ( Parabathynellidae Family), and the Bathynellidae species is around 20%. Both families have high genetic diversity. The genetic divergence of COI is relevant at species level, with significant results found within the morphospecies studied. The two new species, P. fideli sp. nov. and P. pirata sp. nov., have a genetic distance of 16.9–17.3% ( Table 1). The genetic distance between specimens of P. fideli sp. nov. from the same population (Pozo del Agua, CO69) varies from 0.2 to 1.9%, and between different but geographically close populations (Pozo del Agua, CO69 and Fuente del Carnero) the genetic distance is 0.7–1.9% ( Table 1). Nevertheless, the genetic divergence between this new species and the specimens of CO209 cave (also geographically close) ( P. cf. fideli designated in Table 4) varies from 6.5 to 7.9%, which is a very high divergence between populations of the same species. The distance between specimens of P. pirata sp. nov. from the same population (Río Chico) in general ranged from 0.0 to 0.2%, although in the case of five specimens ( P. cf. pirata designated in Table 4) from that same population genetic divergences ranging from 3.7 to 4.3% were seen. This is also a very high divergence between specimens of the same population.

Phylogenetic analyses

The results of the phylogenetic analyses (ML, Bayesian) are summarized in Figure 7. The COI mtDNA sequence data analysis produced a tree in which all samples of Bathynellidae are clearly separated from Iberobathynella burgalensis (Parabathynellidae) . There are two groups supported in ML and Bayesian analyses (100% Bayesian posterior probability and 100% bootstrap values). The phylogenetic reconstruction revealed that the major clades clearly grouped into at least two different species: Paradoxiclamousella fideli gen. nov. sp. nov. and Paradoxiclamousella pirata sp. nov. The first robust group (100% bootstrap) is formed by populations of the P. fideli sp. nov. from CO69 cave and the population of CO209 cave, which appears in a different clade. The other clade is formed by all specimens of the populations from Río Chico cave, but with four specimens of the well-identified lineage of P. pirata sp. nov. in one group, and the other five specimens in another.

The type localities of the two new species identified morphologically as P. fideli sp. nov. and P. pirata sp. nov., are geographically far apart and based on the phylogeny built only with the mitochondrial gene COI, these populations show enough genetic distance to be separated into two completely different clades (see Table 1).

Figures

Figure 1. Paradoxiclamousella fideli sp. nov. (A–D, F–H) Male holotype. (A) Antennule (dorsal view); (B) antenna (dorsal view); (C) paragnath; (D) mandible; (E) mandible female; (F) masticatory part of mandible; (G) maxillule; (H) maxilla. Scale bar in mm.

Figure 2. Paradoxiclamousella fideli sp. nov. (A–G) Male holotype. (A) Thoracopod I; (B) thoracopod II; (C) thoracopod III; (D) thoracopod IV; (E) thoracopod V; (F) thoracopod VI; (G) thoracopod VII. Scale bar in mm.

Figure 4. Paradoxiclamousella pirata sp. nov. (A–G) Male holotype. (A) Antennule (dorsal view); (B) antenna (dorsal view); (C) paragnathes; (D) mandible; (E) masticatory part of mandible; (F) maxillule; (G) maxilla. Scale bar in mm.

Figure 5. Paradoxiclamousella pirata sp. nov. (A–G) Male holotype. (A) Thoracopod I; (B) thoracopod II; (C) thoracopod III; (D) thoracopod IV; (E) thoracopod V; (F) thoracopod VI; (G) thoracopod VII. Scale bar in mm.