Several recent estimates of global biodiversity have concluded that the total number of species on Earth lies near the lower end of the wide range touted in previous decades. However, none of these recent estimates formally explore the real ‘elephant in the room’, namely, what proportion of species are taxonomically invisible to conventional assessments, and thus, as undiagnosed cryptic species, remain uncountable until revealed by multi-gene molecular assessments. Here we explore the significance and extent of so-called ‘hyper-cryptic’ species complexes, using the Australian freshwater fish Galaxias olidus as a proxy for any organism whose taxonomy ought to be largely finalized when compared to those in little-studied or morphologically undifferentiated groups. Our comprehensive allozyme (838 fish for 54 putative loci), mtDNA (557 fish for 605bp of cytb), and morphological (1963–3389 vouchers for 17–58 characters) assessment of this species across its broad geographic range revealed a 1500% increase in species-level biodiversity, and suggested that additional taxa may remain undiscovered. Importantly, while all 15 candidate species were morphologically diagnosable a posteriori from one another, single-gene DNA barcoding proved largely unsuccessful as an a priori method for species identification. These results lead us to draw two strong inferences of relevance to estimates of global biodiversity. First, hyper-cryptic complexes are likely to be common in many organismal groups. Second, no assessment of species numbers can be considered ‘best practice’ in the molecular age unless it explicitly includes estimates of the extent of cryptic and hyper-cryptic biodiversity.
Adams_etal_Syst_Biol_unique_cytb_haplotypes
MEGA file of n=277 unique cytb haplotypes (271 ingroup and 6 outgroup sequences for 602bp),
Adams_etal_Syst_Biol_Supplemental_Table_1
TABLE 1. Locality, collection and sample size information for mountain galaxias specimens used in the molecular studies or prepared and assessed for the morphological studies. Sites arranged according to species, Drainage Division (DD) and River Basin (RB) (AWRC 1976). Alloz = Allozymes; Cytb = Cytochrome-b sequenced; Morph – Morphometrics; Meris – Meristics. Sample sizes for each morphological assessment include the individuals used in the final analyses, plus outliers, plus those subsequently used to assess the geographic range of each species. Taxon codes follow Table 1 and Figure 5. Where provided, sites codes match those of Raadik (2011). Sites were assigned to three regions for some of the allozyme PCoAs, namely ‘northern’ (all sites within DD 2, RB 3–17 or DD 4, RB 10–22), ‘central’ (all sites within DD 2, RB 18–35 or DD 4, RB 1–12), or ‘western’ (all sites within DD 4, RB 26 or DD 5).
Adams_etal_Syst_Biol_Supplemental_Table_2_allozyme_raw_data
Individual allozyme genotypes for n=838 mountain galaxias at 54 allozyme loci. Gel# = individual allozyme code; Taxon/DD/RB/site no = as per Supplemental Table 1; Locus abbreviations as outlined in main text.
Adams_etal_Syst_Biol_Supplemental_Table_3
TABLE 3. Allozyme frequencies for the 15 diagnosable candidate species, as identified using stepwise PCoA. Alleles with a frequency of less than 0.5 % in any taxon are not shown. For polymorphic loci, the frequencies of all but the rarer/rarest alleles are expressed as percentages and shown as superscripts (allowing the frequency of each rare allele to be calculated by subtraction from 100 %). Alleles not separated by a comma all shared the frequency indicated. Taxon codes follow Table 1 and Figure 3. A dash indicates no genotype was assignable at this locus.
Adams_etal_Syst_Biol_Supplemental_Table_4
TABLE 4. Total number of diagnostic ‘characters’ among the 15 candidate species across both the allozyme and morphological datasets. The morphometric and meristic datasets were each only counted as one ‘super-character’, based on the outcomes of our multivariate analyses. In all cases, multiple univariate morphological characters were able to differentiate pairwise combinations of taxa (Raadik 2011).
Adams_etal_hyper-cryptic_olidus_Syst_Biol_Supplementary_Figures_R1
Powerpoint file containing Supplemental Figures 1-6. Each figure is individually captioned.
Adams_etal_hyper-crpytic_olidus_Syst_Biol_Supplementary_Text
Supplementary Text 1 and 2, providing additional details of the morphological analyses undertaken.