Biodiversity inventory of the grey mullets (Actinopterygii: Mugilidae) of the Indo‐Australian Archipelago through the iterative use of DNA‐based species delimitation and specimen assignment methods

Abstract

DNA barcoding opens new perspectives on the way we document biodiversity. Initially proposed to circumvent the limits of morphological characters to assign unknown individuals to known species, DNA barcoding has been used in a wide array of studies where collecting species identity constitutes a crucial step. The assignment of unknowns to knowns assumes that species are already well identified and delineated, making the assignment performed reliable. Here, we used DNA‐based species delimitation and specimen assignment methods iteratively to tackle the inventory of the Indo‐Australian Archipelago grey mullets, a notorious case of taxonomic complexity that requires DNA‐based identification methods considering that traditional morphological identifications are usually not repeatable and sequence mislabeling is common in international sequence repositories. We first revisited a DNA barcode reference library available at the global scale for Mugilidae through different DNA‐based species delimitation methods to produce a robust consensus scheme of species delineation. We then used this curated library to assign unknown specimens collected throughout the Indo‐Australian Archipelago to known species. A second iteration of OTU delimitation and specimen assignment was then performed. We show the benefits of using species delimitation and specimen assignment methods iteratively to improve the accuracy of specimen identification and propose a workflow to do so.

Revisiting the Diversity of Barbonymus (Cypriniformes, Cyprinidae) in Sundaland Using DNA-Based Species Delimitation Methods

“…The present study provides an update of the Barbonymus diversity in Sundaland through the aggregation of newly generated and recently published DNA barcode records, resulting in a reference library consisting of 173 sequences largely distributed across mainland Asia and Sundaland (the haplotype MK978151 was observed in five individuals in [16], resulting in a dataset in BOLD consisting of 169 sequences). DNA-based species delimitation methods largely agreed on the delineation of six MOTUs, except mGMYC with a much higher estimate, a fact that was already reported in other studies [22,27,46,48,60]. Aside from this noticeable exception, methods were concordant in their delimitations, and a barcoding gap was observed between all six MOTUs, with maximum K2P distances mostly <1% within MOTUs, thereby falling into previously observed ranges for Cypriniformes in Sundaland [9,16,21,22].…”

“…Barbonymus collingwoodii (Günther, 1868) and B. mahakkamensis (Ahl, 1922) are endemic to North and East Borneo, respectively, and B. belinka (Bleeker, 1860) is an endemic species of the west coast of Sumatra. DNA barcoding, the use of cytochrome oxidase I (COI) as a species tag for automated identification, opened new perspectives for the characterization of Sundaland's ichthyofauna by helping to clarify taxonomic confusion within several groups [15,21,22], by identifying discrepancies in historical species records [9] and by detecting a substantial amount of morphologically similar, yet highly divergent lineages (i.e., cryptic diversity) within numerous species [9,15,[21][22][23][24][25][26][27][28]. Several molecular studies that aimed at characterizing patterns of genetic diversity in Barbonymus led to conflicting species identities associated with sequences submitted to international repositories [9,16,[29][30][31][32][33][34][35][36].…”

Integrative taxonomy reveals extreme morphological conservatism in sympatricMugilspecies from the Tropical Southwestern Atlantic

“…Molecular variation in the COI gene has proven to be a powerful tool to resolve complicated phylogenetic relationships, estimate divergence times, and identify mislabelled specimens in Mugil (Delrieu‐Trottin et al., 2020; Durand, Chen, Shen, Fu, & Borsa, 2012; Durand et al., 2017; Neves et al., 2020). Thus, to identify evolutionary lineages, we performed a Bayesian phylogenetic analysis with posterior probabilities (PP) based on the COI gene.…”

“…We then performed Sanger sequencing only for the forward strand. This fast‐evolving gene is often used to diagnose species (Hebert, Ratnasingham, & Waard, 2003) and has been shown to clearly distinguish cryptic species of Mugil (Delrieu‐Trottin et al., 2020; Durand, Hubert, Shen, & Borsa, 2017). All 129 sequences were edited with BioEdit v.3.3.19 (Hall, 1999) and then translated into amino acids to verify the lack of stop codons.…”

Assessing fish diversity of Merbok Estuary and adjacent marine waters by DNA taxonomy: towards building comprehensive DNA barcode reference library

“…Our results are consistent with this study, further indicating that differentiation in this species is not only allopatrically but, also, sympatrically distributed. Recent molecular taxonomic studies on the family Mugilidae in which are included O. perusii and P. subviridis, evidenced a very high level of cryptic diversity in the Indo-West Paci c region 46,47 . Several valid mullet species are actually complexes of several morphologically similar species for which extensive taxonomic revisions are needed.…”