Resolving Cryptic Species of Bossiella (Corallinales, Rhodophyta) Using Contemporary and Historical DNA 1
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RESEARCH ARTICLE AMERICAN JOURNAL OF BOTANY Resolving cryptic species of Bossiella (Corallinales, Rhodophyta) using contemporary and historical DNA 1 Katharine R. Hind 2,3,6 , Kathy Ann Miller4 , Madeline Young2 , Cassandra Jensen2 , Paul W. Gabrielson 5 , and Patrick T. Martone 2,3 PREMISE OF THE STUDY: Phenotypic plasticity and convergent evolution have long complicated traditional morphological taxonomy. Fortunately, DNA sequences provide an additional basis for comparison, independent of morphology. Most importantly, by obtaining DNA sequences from historical type specimens, we are now able to unequivocally match species names to genetic groups, often with surprising results. METHODS : We used an integrative taxonomic approach to identify and describe Northeast Pacif c pinnately branched species in the red algal coralline genus Bossiella , for which traditional taxonomy recognized only one species, the generitype, Bossiella plumosa. We analyzed DNA sequences from histori- cal type specimens and modern topotype specimens to assign species names and to identify genetic groups that were dif erent and that required new names. Our molecular taxonomic assessment was followed by a detailed morphometric analysis of each species. KEY RESULTS: Our study of B. plumosa revealed seven pinnately branched Bossiella species. Three species, B. frondescens , B. frondifera , and B. plumosa , were assigned names based on sequences from type specimens. The remaining four species, B. hakaiensis , B. manzae , B. reptans , and B. montereyensis , were described as new to science. In most cases, there was signif cant overlap of morphological characteristics among species. CONCLUSIONS: This study underscores the pitfalls of relying upon morpho-anatomy alone to distinguish species and highlights our likely underestimation of species worldwide. Our integrative taxonomic approach can serve as a model for resolving the taxonomy of other plant and algal genera. K E Y W O R D S Bossiella ; COI-5P; coralline algae; Corallina frondescens ; cryptic species; psbA ; rbcL ; integrative taxonomy T e process of species identif cation and description has changed variants of a single species (e.g., Gabrielson et al., 2011 ; Hind et al., dramatically over the last 50 years. Traditionally, taxonomists re- 2014a ), and genetically distinct species sometimes exhibit nearly lied solely on morpho-anatomical criteria to delineate species. identical morphologies (e.g., van der Merwe et al., 2015 ). More re- However, we now know that this approach can be problematic due cently, the incorporation of molecular sequence data, particularly to high levels of intraspecif c variation in morphological characters DNA barcodes (standardized regions of DNA), into taxonomic re- resulting from phenotypic plasticity ( Stewart, 2006 ; Koehl et al., search has achieved more accurate estimates of species diversity 2008 ; Monro and Poore, 2009 ) and convergent morphologies be- and at less cost than traditional morphological methods ( Hebert tween distantly related species due to analogous environmental et al., 2003 , 2004 ; T ompson and Newmaster, 2014 ). A growing pressures ( Johansen, 1981 ; Steneck, 1986 ). For example, morpho- trend in diversity studies is to use integrative approaches ( Padial logically distinct specimens have turned out to ref ect morphological et al., 2010 ), such as molecular-assisted alpha taxonomy (MAAT) ( Saunders, 2005 , 2008 ; Cianciola et al., 2010 ), that combine molec- ular and classic morpho-anatomical data to identify and describe 1 Manuscript received 30 June 2015; revision accepted 29 September 2015. organisms. 2 Department of Botany and Biodiversity Research Centre, University of British Columbia, 6270 University Blvd., Vancouver, British Columbia, Canada, V6T 1Z4; Af er species have been delineated, taxonomists are challenged 3 Hakai Institute, Pruth Harbour, Calvert Island, British Columbia, Canada V0P 1H0; with applying names to these species. Naming botanical species is 4 University Herbarium, Silva Center for Phycological Documentation, 1001 Valley Life governed by the International Code of Nomenclature for Algae, Sciences Building #2465, University of California, Berkeley, California 94720 USA; and Fungi and Plants ( McNeill et al., 2012 ) and is based on a “type” 5 Department of Biology and Herbarium, 3280 Coker Hall, University of North Carolina, Chapel Hill, North Carolina 27599 USA system of nomenclature. T is task involves an analysis of the type 6 Author for correspondence (e-mail: [email protected]) specimen, the specimen to which the name is permanently attached doi:10.3732/ajb.1500308 ( McNeill et al., 2012 , Article 7.2), and an exploration of all available AMERICAN JOURNAL OF BOTANY 102 ( 11 ): 1 – 19 , 2015 ; http://www.amjbot.org/ © 2015 Botanical Society of America • 1 2 • A M E R I C A N J O U R N A L O F B O T A N Y species names that might be applied to a genetic group or clade. specimen of Corallina frondescens Postels & Ruprecht showed that Sometimes type specimens are inaccessible, missing, or unavailable this species also belongs in Bossiella. for anatomical investigation and/or DNA extraction. Consequently, Previously, Hind et al. (2014b) reassessed those Bossiella s p e c i e s in place of the type specimen, topotype material (contemporary with dichotomous branching passing under the names B. califor- specimens from the collection site of the type specimen) is of en nica (Decaisne) P.C.Silva and B. orbigniana (Decaisne) P.C.Silva. used as a surrogate, even though type locality information can be Future studies will assess those species with irregular branching, vague and environmental conditions may have changed dramati- falling under the name B. chiloensis (Decaisne) H.W.Johansen. cally since the original collection was made. For example, anthro- pogenic changes in the environment such as f sh-farming or coastal development may increase algal abundance ( Sanderson et al., 2012 ) MATERIALS AND METHODS or decrease algal diversity ( Bates et al., 2009 ). In addition, herbar- ium records from the last 70 years documented signif cant pole- Sample collection — T is work represents a collaborative ef ort ward shif s of seaweed species in Australia ( Wernberg et al., 2011 ). among many laboratories whose goal was to conduct f oristic sur- T us, returning to the original type locality to collect specimens veys of algae along the Northeast (NE) Pacif c coast from Alaska, does not guarantee the presence of the target species. United States through Baja California, Mexico using an integrative Another technique to apply species names to contemporary col- taxonomic approach. Together, we analyzed morphological and se- lections is the use of historical DNA sequencing. Historical DNA quence data for 331 Bossiella specimens, including both contempo- approaches have been used extensively across many taxa including rary and historic collections. Specific collection localities, GPS woolly mammoths ( Hagelberg et al., 1994 ) and algal fossils ( Hughey coordinates, and collector information are listed in Appendix S1 et al., 2008 ). Adoption of these techniques has come with some ap- (see Supplemental Data with the online version of this article). prehension as to the reliability of these data due to contamination Efforts were made to obtain topotype material from type locali- and proper experimental controls ( Saunders and McDevit, 2012 ). ties (Appendix S1). Multiple specimens of each currently recog- However, these issues have been addressed ( Hughey and Gabrielson, nized Bossiella species in the NE Pacific ( Gabrielson et al., 2012 ; 2012 ), and sequences from type specimens have been essential to H i n d e t al., 2014b ), i.e., B. californica, B. chiloensis, B. dichotoma apply names to contemporary collections of morphologically vari- (Manza) P.C.Silva, B. heteroforma K.R.Hind, P.W.Gabrielson & able genera ( Hughey et al., 2001 ; Gabrielson, 2008 ; Lindstrom et al., G.W.Saunders, B. orbigniana , B. plumosa (Manza) P.C.Silva, and 2011 , 2015 ; Hind et al., 2014b ). B. schmittii (Manza) H.W.Johansen were made at each collection In this paper, we used both topotype collections and historical locality. Specimens were collected from the high, mid, and low in- DNA approaches to apply names to contemporary specimens in a tertidal zones and subtidally to depths of 20 m (Appendix S1). A group of red algae with known taxonomic uncertainties. T e Coral- portion (3 × 3 cm) of each collection was silica dried for DNA anal- linales is a large order (over 700 species) of calcif ed red algae that yses, and the remaining material was dried as a voucher and acces- are notoriously dif cult to identify due to convergent and simple sioned at UBC, UNB, NCU or UC (Appendix S1; see T iers (2015 ) morphologies ( Hind et al., 2014a ; Pardo et al., 2014 ). T ey are im- for herbarium acronyms). portant components of marine ecosystems, acting as ecosystem en- Type collections were observed and photographed, and tissue gineers ( Asnaghi et al., 2015 ), providing structural support to reefs samples (intergenicula) were excised for genetic analyses (Appen- ( Nelson, 2009 ) and emitting chemical settlement cues to marine dix S1). Specimens from the following herbaria were examined: invertebrate species ( Whalan et al., 2012 ), yet we know little about TRH, S, UBC, and UC. their specif c identities. T e order comprises species with upright, usually jointed fronds (geniculate species) and prostrate crusts DNA extraction and PCR — All contemporary samples were