DOI: 10.5061/dryad.0k6djh9wp
title: How did subterranean amphipods cross the Adriatic Sea? Phylogenetic evidence for the dispersal-vicariance interplay mediated by marine regression-transgression cycles

description: Aim: We tested the hypothesis that historical marine
 regression-transgression cycles shaped the distribution patterns of
 subterranean amphipods through repeated cycles of dispersal and vicariance
 against the hypothesis that subterranean amphipods colonized both sides of
 the Adriatic Sea independently. Location: Western Balkan Peninsula,
 Adriatic Sea Islands and Apennine Peninsula, Europe Taxon: A clade of
 freshwater subterranean amphipods, genus Niphargus (Crustacea: Amphipoda).
 Methods: The taxonomic structure of the studied clade was revised using
 unilocus species delimitations. Timeframe of cladogenetic events was
 inferred using multilocus time-calibrated phylogeny and compared to the
 main regression-transgression events in Miocene and Pleistocene. The
 geographic origin of the studied clade, species’ range expansions and
 contractions, as well as vicariant events were assessed through modelling
 of historical biogeography. Results: Subterranean amphipods of the genus
 Niphargus, found on both sides of the Adriatic Sea, form a monophyletic
 clade. The reconstructions of ancestral ranges suggest that the clade
 emerged in the Balkan Peninsula, three times independently dispersed to
 the Apennine Peninsula and once back to the Balkans. Adriatic Islands were
 colonized multiple times, predominantly from the Balkan Peninsula. The
 dispersal-vicariance events correspond to historical
 regression-transgression cycles in Miocene and Pleistocene. Main
 conclusions: Marine regression-transgression cycles shaped distribution
 patterns of subterranean amphipods, while the alternative hypothesis
 received no support. The subterranean faunas apparently well reflect older
 biogeographic events.
description: <i>Taxon sampling and
 DNA isolation</i> Taxon sampling aimed to include a
 wide range of <i>Niphargus </i>species, distributed from
 Ireland to Iran, and to comprehensively sample species distributed on the
 both sides of the Adriatic Sea. We sampled altogether 214 localities and
 totally 494 individuals, and stored them in 96% ethanol.  Each specimen was identified to
 the closest morphologically resembling morphospecies using original
 species descriptions.  <i>Taxonomic structure and
 molecular species delimitation</i> In order to account for
 incomplete taxonomy and the possible presence of morphologically cryptic
 species, we revised the taxonomic structure of the study dataset prior to
 the main analyses. We assembled a dataset containing altogether 463 COI
 sequences of <i>Niphargus</i> and genera nested within it
 (<i>Carinurella, Haploginglymus, Niphargobates,
 Pontoniphargus</i>) The sequences were acquired in this study or
 retrieved from GenBank. Putative species structure, represented by the so
 called molecular taxonomic operational units (hereafter MOTUs) was
 inferred using distance- and tree-based delimitation methods, automatic
 bridge gap discovery (hereafter ABGD) (Puillandre, Lambert, Brouillet,
 & Achaz, 2012) and Poisson tree processes (hereafter PTP) (Zhang,
 Kapli, Pavlidis, & Stamatakis, 2013). <i>Phylogenetic
 analyses</i> Phylogenetic structure of the
 studied dataset was inferred using 195 Niphargidae species
 (<i>Niphargus</i> and the genera nested within)<i>
 </i>rooted with five species of <i>Pseudoniphargus</i>,
 which was shown to be a sister group in two previous studies
 (Jurado-Rivera et al., 2017; Copilaş-Ciocianu et al., 2020). The dataset
 included 169 molecular operational taxonomic units (MOTUs) delimited in
 the PTP analysis (see above and Results section) supplemented with 31
 described species. Phylogenetic relationships were reconstructed using two
 alternative approaches: i) maximum likelihood with partition-specific
 setting and ultrafast bootstrapping in IQ-tree 1.6.7 (Nguyen, Schmidt, Von
 Haeseler, & Minh, 2015), and ii) Bayesian inference with
 partition-specific settings in MrBayes 3.2.6 (Ronquist et al.,
 2012). <i>Estimation of divergence
 times</i> In order to define the timeframe
 of splits between the lineages distributed in the Dinaric Karst and the
 Apennine Peninsula, we reconstructed a time-calibrated multilocus
 phylogeny using the package BEAST 2.5.1 (Bouckaert et al. 2018). The
 dataset comprising 200 individuals and four molecular markers was analysed
 using partitioning schemes with distinct parameters settings, following
 best-fit models of evolution proposed by bModelTest 1.2.1 (Bouckaert
 & Drummond, 2017). Substitution and clock models were unlinked for
 all partitions, while the tree partitions were linked to generate a single
 dated phylogeny. Birth death and Yule speciation tree priors and their
 marginal likelihoods were compared in BEAST’s extension Path Sampler.
 Based on marginal likelihood, we used a Yule speciation tree prior for the
 final analyses (results not shown). In order to account for
 lineage-specific rate heterogeneity, we employed a lognormal relaxed clock
 (Drummond, Ho, Phillips, & Rambaut, 2006).  <i>Modelling past
 dispersal, vicariance and reconstruction of ancestral
 ranges</i> In order to elucidate the
 historical biogeography of the main Transadriatic clade, we modelled
 dispersal, extinction, vicariance, cladogenesis and ancestral ranges (Fig.
 1) using package BioGeoBEARS 1.1.2 (Matzke, 2013) in R 3.5.3 (R Core Team,
 2019).