Phylogeny and species diversity of Tasmanian mountain shrimps and their relatives (Crustacea, Anaspidesidae)

Abstract

Anaspidacea is an enigmatic taxon within Malacostraca with uncertain phylogenetic affinities. One of the four families within Anaspidacea, the Anaspidesidae (representing in most respects the plesiomorphic conditions of the entire taxon), is endemic to Tasmania and shows a remarkable geographical and vertical distribution. Eleven species in three genera (Allanaspides, Anaspides and Paranaspides) are recognized to date, occurring in freshwater habitats such as lakes, rivers, streams, tarns and also in caves. We present the most detailed analyses to date of the phylogeny of Anaspidesidae including all eleven species based on molecular data (COI, 16S and 28S). In addition, the genetic differentiation within the morphologically recognized species is studied and a dating of speciation is given using a molecular clock approach (based on mt-DNA only). A special focus is on the multiple instances of immigration into the subterranean habitat. Most of the morphologically delineated species are recognized by molecular data. Significantly, however, in at least two cases, the molecular data suggest more species than so far described, but further molecular sampling from a wider range of localities is required. Our molecular divergence estimates suggest the anaspidesids are younger than previously thought. Rather than having Eocene origins, our results suggest Early Miocene origins with primary diversification in Miocene and Pliocene. Entry into subterranean habitats appears to have occurred in the Pleistocene rather than Tertiary. Thus, rather than being a defensive response to rising temperatures in the Tertiary, entry into caves in the Pleistocene may represent opportunistic expansion into new habitats created by the action of glacial meltwaters on existing karst formations. As a result, environmental changes during the Pleistocene appear to have prompted ecological expansion of Anaspides by "opening" new subterranean habitats leading to increased diversification in the group. K E Y W O R D Scaves, phylogeography, subterranean habitat, Syncarida

Genetic structure and new occurrence records of the iconic Tasmanian mountain shrimp

“…Finally, A. tasmaniae, formerly believed to be widespread over the whole island in surface waters and caves, now appeared to be limited to a small range on the east and south-east side of Mt Wellington above 450 m asl with no known cave occurrences. Richter et al (2018), using molecular data, largely corroborated the major species clades of Ahyong (2016) and recovered a monophyletic A. tasmaniae, with different sister group relationships depending on the gene fragment used.…”

“…In total, 37 specimens of A. tasmaniae were collected and newly sequenced for this study (see Table S1, Supplementary Material). In addition, a total of 9 COI, 11 16S rRNA and one 28S rRNA sequence from the study by Richter et al (2018) were included. The newly sequenced specimens were collected using hand nets and preserved directly in 80-95% ethanol.…”

“…The polymerase chain reaction (PCR) had a total volume of 30 mL containing 3 mL of each primer (each 10 mM), 3 mL 10 Â buffer (Sigma Aldrich), 3 mL dNTP mix (2 mM, Biozym), 2.58 mL MgCl 2 (25 mM, Sigma Aldrich), 0.12 mL Taq-polymerase (Sigma Aldrich), 10.71 mL ultrapure water and 4.5 mL of the DNA extract. Primers were LCO2 (5 0 -TCN ACH AAY CAT AAA GAY ATT GGA AC-3 0 ) (Richter et al 2018), HCO2198 (5 0 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 0 ) (Folmer et al 1994) for COI, 16Sa (5 0 -CGC CTG TTT ATC AAA AAC AT-3 0 ), 16Sb (5 0 -CTC CGG TTT GAA CTC AGA TCA-3 0 ) (Xiong and Kocher 1991) for 16S and rd4.8a (5 0 -ACC TAT TCT CAA ACT TTA AAT GG-3 0 ) and rd7b1 (5 0 -GAC TTC CCT TAC CTA CAT-3 0 ) (Richter et al 2007) for 28S rDNA. PCR amplification programs comprised an initial denaturation step at 94 C for 1 min, followed by 40 amplification cycles (94 C for 30 s, 50 C for 30 s, 72 C for 60 s) for COI, 38 amplification cycles (94 C for 30 s, 48 C for 30 s, 72 C for 60 s) for 16S, 35 amplification cycles (94 C for 30 s, 55 C for 30 s, 72 C for 60 s) for 28S, and a final elongation step at 72 C for 5 min.…”

A new species of Tasmanian mountain shrimp, Anaspides driesseni sp. nov. (Malacostraca, Anaspidacea, Anaspidesidae)

“…Moreover, the monophyly of A. driesseni and its position as sister species to A. swaini form 1 are supported by mitogenomic analysis of the genus (Höpel et al, unpublished). On the other hand, in A. swaini form 1 as now restricted, the high intraspecific distances (up to 10.3 % in COI) are striking, especially between the lineages found in tributaries of the Weld River (Richter et al, 2018). Nevertheless, these lineages are sympatric and no significant morphological differences between these lineages were detectable.…”

“…The polymerase chain reaction (PCR) had a total volume of 30 μl which contained 3 μl of each primer (each 10 μM), 3 μl 10× buffer (Sigma Aldrich), 3 μl dNTP mix (2 mM, Biozym), 2.58 μl MgCl 2 (25 mM, Sigma Aldrich), 0.12 μl Taq-polymerase (Sigma Aldrich), 10.71 μl ultrapure water and 4.5 μl of the DNA extract. Primers were LCO2 5'-TCN ACH AAY CAT AAA GAY ATT GGA AC-3' (Richter et al, 2018), HCO2198 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3' (Folmer et al, 1994) for COI, 16Sa 5'-CGC CTG TTT ATC AAA AAC AT-3', 16Sb 5'-CTC CGG TTT GAA CTC AGA TCA-3' (Xiong & Kocher, 1991) for 16S and rd4.8a 5'-ACC TAT TCT CAA ACT TTA AAT GG-3', rd7b1 5'-GAC TTC CCT TAC CTA CAT-3' (Richter et al, 2007) for 28S rRNA. PCR amplification programs comprised an initial denaturation step at 94°C for 1 min, followed by 40 amplification cycles (94°C for 30 s, 50°C for 30 s, 72°C for 60 s) for COI, 38 amplification cycles (94°C for 30 s, 48°C for 30 s, 72°C for 60 s) for 16S, 35 amplification cycles (94°C for 30 s, 55°C for 30 s, 72°C for 60 s) for 28S and a final elongation step at 72°C for 5 min.…”