A Broad Phylogenetic Analysis of Boraginaceae: Implications for the Relationships of Mertensia

The phylogenetic relationships of Mertensia (Boraginaceae), which comprises approximately 45 species in both Asia and North America, have been uncertain, and taxonomists have placed the genus in various tribes of subfamily Boraginoideae, with the most recent placements in Trigonotideae and Cynoglosseae. Our study applies molecular phylogenetic methods to test the monophyly and relationships of Mertensia. We used DNA sequence data from the nuclear ribosomal nrITS region and four cpDNA regions (matK, ndhF, rbcL, trnL-trnF) to examine the placement of Mertensia among a sampling of accessions from approximately 70% of the genera of Boraginaceae s. l. Phylogeny reconstructions using maximum parsimony, maximum likelihood, and Bayesian inference were largely congruent with previous molecular phylogenetic analyses of Boraginaceae that had applied far fewer taxa. We recovered five deep clades that correspond to Boraginaceae subfamilies Boraginoideae, Cordioideae, Heliotropioideae, Hydrophylloideae, and Ehretioideae (including Lennoa and Pholisma). In subfamily Boraginoideae, we recovered clades that correspond to the tribes Echiochilieae, Lithospermeae, Cynoglosseae, and Boragineae, although several tribes previously circumscribed on the basis of morphological data were not recovered as monophyletic in our results. Based on the sister relationship between the genus Codon and subfamily Boraginoideae found in our phylogeny reconstructions, we propose Codoneae as a new tribe of Boraginoideae. We recovered strong support for the monophyly of Mertensia and the placement of the monotypic genus Asperugo as its sister. Mertensia and Asperugo were strongly supported as members of Cynoglosseae. Keywords—Asteridae, Boraginaceae, Boraginoideae, maximum likelihood, Mertensia, molecular phylogenetics. Mertensia Roth (Boraginaceae, subfamily Boraginoideae) comprises approximately 45 species distributed in the Northern Hemisphere of both North America and Asia (Williams 1937; Popov 1953a; Al-Shehbaz 1991). Mertensia shares many characteristics with other Boraginoideae, including hispid to strigose vestiture of stout, eglandular, unicellular trichomes, pentamerous sympetalous corolla bearing faucal appendages, and fruit a schizocarp of four one-seeded nutlets (Al-Shehbaz 1991). Mertensia is distinguished from other members of the Boraginoideae by undivided stigmas and a ventral and suprabasal (attached above the base) position of the oblique attachment scar of abscised nutlets (Johnston 1924a; Popov 1953b; Al-Shehbaz 1991). However, it is uncertain whether the stigma form or the nutlet attachment scar states are synapomorphies for the genus. Since first described by Linnaeus (1753), Mertensia has been variously assigned to five different tribes within subfamily Boraginoideae: Symphyteae, Lithospermeae, Eritrichieae, Trigonotideae, and Cynoglosseae (Table 1). Its early placement in Symphyteae (Don 1838, p. 307) was based on the presence of a tubular corolla, included stamens, and “carpels or nuts fixed to the bottom of the calyx.” This latter observation that the nutlets are adnate to the calyx is morphologically unlikely, given that the flowers are hypogynous and have the gynoecium separated from the calyx by an encircling corolla. Mertensia was later placed in Lithospermeae (De Candolle 1846; Bentham 1876; Baillon 1890; Gürke 1897), but this was questioned by Johnston (1924a, 1924b), who placed it in Eritrichieae, based on nutlet attachment and simple stigmas. In contrast, Popov (1953b) placed Mertensia in the subtribe Trigonotidinae of Lithospermeae, stating that the subtribe was intermediate between Eritrichieae and typical Lithospermeae. Riedl (1967, 1968) disagreed with Popov’s placement of Mertensia in Lithospermeae and included both Mertensia and Trigonotis Stev. in subtribe Trigonotidinae of the newly recognized tribeTrigonotideae. (BothPopov andRiedl denoted the subtribe Trigonotidinae erroneously as “Trigonotideae.” Although the Riedl and Popov subtribe name was adopted by subsequent workers, we follow the International Code of Botanical Nomenclature and use subtribe Trigonotidinae to denote this group in our study). Recent investigations of the phylogenetic relationships of Trigonotideae using molecular data suggest the tribe is polyphyletic and that Mertensia resides as a distinct lineage deeply nested in the tribe Cynoglosseae (Weigend et al. 2010). Problems with the tribal placement of Mertensia are exacerbated by uncertainty about its closest relatives. Several authors have suggested relationships to other borage genera on the basis of morphological characters. For example, Johnston (1924b) suggested Anoplocaryum Ledeb., a genus of about five species in Asia (Nasir 2006), was closely allied to Mertensia, with the two genera differing only in corolla shape and nutlet attachment. Popov (1953b) suggested the monotypic genus Brachybotrys Maxim. ex Oliv. was most closely related to Mertensia. Al-Shehbaz (1991) asserted Trigonotis was closely related to Mertensia, from which it differed in flower size, corolla shape, and nutlet shape. Recent molecular studies based on chloroplast and nuclear data have inferred close relationships with other genera. For example, Mertensia was placed as sister to Lappula Moench and Hackelia Vasey ex Beal in Olmstead and Ferguson (2001; Olmstead, pers. comm.). In contrast, Mansion et al. (2009) inferred a sister relationship toOmphalodesMill. More recently, Weigend et al. (2010) found Mertensia to be sister to a clade consisting of Eritrichium Schrad. ex Gaudin, Hackelia, and Lappula, but this relationship had limited support. These varying placements of Mertensia in molecular phylogenetic studies may reflect the different taxon sampling of each study as well as the relatively few borage genera sampled. For example, the three studies that have sampled the most borage genera (Långström and Chase 2002; Mansion et al. 2009; Weigend et al. 2010) included no more than 40 of the approximately 150 genera within Boraginaceae (sensu APG 2003, 2009). The limited sampling of putative close relatives