A new large-flowered species of Andeimalva... 91 A new large-flowered species of Andeimalva (Malvaceae, Malvoideae) from Peru Laurence J. Dorr1, Carolina Romero-Hernández2, Kenneth J. Wurdack1 1 Department of Botany, MRC-166, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, Washington, D.C. 20013-7012, USA 2 Missouri Botanical Garden Herbarium, William L. Brown Center, P.O. Box 299, Saint Louis, MO 63166-0299, USA Corresponding author: Laurence J. Dorr (dorrl@si.edu) Academic editor: Clifford Morden  |  Received 28 August 2018  |  Accepted 11 October 2018  |  Published 5 November 2018 Citation: Dorr LJ, Romero-Hernández C, Wurdack KJ (2018) A new large-flowered species of Andeimalva (Malvaceae: Malvoideae) from Peru. PhytoKeys 110: 91–99. https://doi.org/10.3897/phytokeys.110.29376 Abstract Andeimalva peruviana Dorr & C.Romero, sp. nov., the third Peruvian endemic in a small genus of five species, is described and illustrated from a single collection made at high elevation on the eastern slopes of the Andes. Molecular phylogenetic analyses of nuclear ribosomal ITS sequence data resolve a group of northern species of Andeimalva found in Bolivia and Peru from the morphologically very different south- ern A. chilensis. The new species bears the largest flowers of any Andeimalva and is compared with Bolivian A. mandonii. A revised key to the genus is presented. Keywords Andeimalva, Andes, Malvaceae, Malvoideae, Peru, phylogeny Introduction The genus Andeimalva J.A. Tate (Malvaceae, Malvoideae) was created to accommo- date four species found in the Andes of South America from northern Peru to central Chile and includes three species previously placed in Tarasa Phil. and one in Mala- cothamnus Greene (Tate 2003). Krapovickas (1965) described two of these species, T. machupicchensis Krapov. and T. spiciformis Krapov., and allied them with a third, PhytoKeys 110: 91–99 (2018) doi: 10.3897/phytokeys.110.29376 http://phytokeys.pensoft.net Copyright Laurence J. Dorr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Launched to accelerate biodiversity research A peer-reviewed open-access journal Laurence J. Dorr et al. / PhytoKeys 110: 91–99 (2018)92 T. mandonii (Baker f.) Kearney, because he observed that the three differed from all other species of Tarasa in having entire lanceolate leaves more than three times as long as wide and awned mericarps with smooth (versus reticulate) sides. Despite the distinctiveness of this group, Krapovickas (1965) chose not to recognize it formally. The fourth species of Andeimalva, endemic to Chile, was long known as M. chilensis (Gay) Krapov. even though its placement in Malacothamnus was problematic based on its morphology, cytology, and geography (Bates 1963; Bates and Blanchard 1970; Tate and Simpson 2003). Tate (2003), combining molecular, cytological and mor- phological data, transferred these three species of Tarasa and one species of Mala- cothamnus to the newly described genus Andeimalva. The Chilean species of Andei- malva is anomalous morphologically in that it differs from the group recognized by Krapovickas (1965) in having suborbicular, slightly 3–5-lobed leaves and awnless mericarps. Nonetheless, molecular and cytological data indicate that it is closely re- lated to the other three species found in Bolivia and Peru (Tate and Simpson 2003). Recent floristic exploration in the northern Andes of Peru yielded a Malvaceae collection (Vega Ocaña 419) of uncertain initial generic affiliation. With further study and molecular phylogenetic analyses, we determined it to represent a new species of Andeimalva, which is described here as A. peruviana Dorr & C.Romero, and the third one endemic to Peru. We are amazed that a shrub with such conspicuous flowers is not known from earlier collections and can only speculate that either its range is very re- stricted in a botanically poorly explored area or it might simply have passed unnoticed due to a limited flowering period. Materials and methods We sought molecular data both to confirm the generic identity of the new collection and to provide a phylogenetic context within the ITS (Internal Transcribed Spacer region of the nuclear ribosomal repeat) data set of Tate and Simpson (2003). Leaf tis- sue with abaxial trichomes scraped off was ground in a bead mill and extracted with the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, California, USA) following an ex- tended digestion with the AP1 buffer emended with 1.0 mg proteinase K (PCR grade solution, Roche Applied Science, Germany) and 1.8% v/v 2-mercaptoethanol. To re- duce systemic contamination risk, all equipment was dedicated to “antique DNA” in a pre-PCR extraction laboratory and no other Malvales were co-extracted or ampli- fied at the same time. Despite being a 2.5-year old collection with reasonably good preservation (i.e., retaining some color and alcohol treated), the resulting extraction only yielded highly degraded DNA (< 400 bp on an agarose test gel). Amplifications of ITS were attempted using a series of nested primers for decreasing amplicon sizes, and only a 302 bp fragment of ITS1 could be recovered with primers 1m (CGTAG- GTGAACCTGCGGAAGGATC, newly modified from ITS1) and P2 (GCCRAGA- TATCCGTTGCCGAG; Cheng et al. 2016). Amplification reactions were conducted in a final volume of 15 µl with 1× Bioline reaction buffer, 8 mM dNTPs, 1.75 mM A new large-flowered species of Andeimalva... 93 magnesium chloride, 2 nM of each primer, 0.1 mg/ml of bovine serum albumin, 0.025 U/µl of Biolase DNA polymerase (Bioline USA, Taunton, Massachusetts, U.S.A.), 1 M betaine, and 1 µl of DNA extraction. Sequencing was with BigDye Termina- tor v3.1 chemistry (Thermo Fisher Scientific, Waltham, Massachusetts) on an ABI 3730xl DNA Analyzer (Thermo Fisher Scientific). The new ITS1 sequence (GenBank MK044847, from Vega Ocaña 419) was manually aligned using a similarity criterion with the Tate and Simpson (2003) data set archived in TreeBASE (http://purl.org/ phylo/treebase/phylows/matrix/TB2:M516). Small changes were made to the source matrix including converting mixed bases to ambiguity codes and improving the align- ment within Fuertesimalva Fryxell (i.e., inconsistencies relative to positions 114–155 in our alignment), which reduced local branch lengths relative to prior analyses. The 53-tip matrix has an aligned length of 696 bp and relatively few, mostly small 1–4 bp indels (except Fuertesimalva spp. sharing a 42 bp deletion); we did not exclude any data in the analyses. We could not obtain any new plastid data and thus did not analyze the plastid data set of Tate and Simpson (2003), which the authors did not combine with ITS due to incongruence. Parsimony (MP) analyses were conducted with PAUP* 4.0a build 163 (Swofford 2002) using 1000 random addition replicates with TBR branch swapping and no search limits, and 1000 bootstrap (MP-BS) replicates, each with two random additions and a search limit of 10,000 trees per iteration (MulTrees = 10,000). Maximum likelihood (ML) analyses were conducted under a GTR + I + Γ model with GARLI 2.01 (Zwickl 2006) on CIPRES XSEDE (https://www.phylo.org/), using 100 replicates for optimal trees, and 1000 ML bootstrap replicates (ML-BS), each with two random additions and the automated stopping criterion. Data resources The data underpinning the analyses reported in this paper are deposited in the Dryad Digital Repository at: https://doi.org/10.5061/dryad.44dm150 Results Suspicions that Vega Ocaña 419 belonged with Andeimalva were confirmed by mo- lecular data where BLAST searches of our ITS1 sequence against GenBank yielded best matches with that genus. This affiliation was confirmed by phylogenetic analyses (Fig. 1) that placed it as strongly supported (BS 95–100) within a nested subclade of Andeimalva, although the exact sister relationship resolved with A. mandonii was weakly supported (ML-BS 75, MP-BS<50). Parsimony analysis yielded a single is- land with 180 trees (length = 346 steps, CI = 0.682 or 0.613, excluding uninforma- tive characters; 128 parsimony informative characters) and a topology similar to prior phylogenies (i.e., Tate 2003; Tate and Simpson 2003). The topology of our ML tree (best score of 100 trees, -lnL = -3137.458) was also similar but not identical to any of Laurence J. Dorr et al. / PhytoKeys 110: 91–99 (2018)94 the parsimony trees. There is a 3-bp deletion (loss of tandem GTT repeat at positions 280–282 in our alignment) only possessed by A. chilensis (Gay) J.A. Tate and A. ma- chupicchensis (Krapov.) J.A. Tate, but this is not reflected in the recovered relationships and would not appear to be a structural synapomorphy. Tarasa geranioides3 Tarasa latearistata1 Tarasa cerrateae1 Tarasa cerrateae2 Tarasa antofagastanaA2 Tarasa antofagastanaC1 Tarasa heterophylla Tarasa antofagastanaA1 Tarasa antofagastanaB Tarasa antofagastanaC2 Tarasa geranioides1 Tarasa pediculata Tarasa corrugata Tarasa tarapacana2 Tarasa urbaniana Tarasa cardenasii Tarasa latearistata2 Tarasa meyeri Tarasa nototrichoides Tarasa tenella Tarasa capitata Tarasa tarapacana1 Tarasa operculata1 Tarasa thyrsoidea1 Tarasa operculata2 Tarasa thyrsoidea2 Nototriche anthemidifolia Nototriche flabellata Nototriche pedicularifolia Tarasa congestiflora Tarasa marinii Tarasa rhombifolia Tarasa geranioides2 Tarasa odonellii Tarasa tenuis Tarasa trisecta Tarasa albertii Tarasa humilis Sphaeralcea crispa Sphaeralcea philippiana Sphaeralcea cordobensis Sphaeralcea angustifolia Sphaeralcea wrightii Andeimalva mandonii Andeimalva peruviana Andeimalva machupicchensis Andeimalva spiciformis Andeimalva chilensis Fuertesimalva jacens Fuertesimalva limensis Urocarpidium albiflorum Modiola caroliniana Modiolastrum lateritium 100/100 96/98 77/73 52/52 100/100 89/85 60/51 82/76 85/82 87/65 59/<50 94/89 97/95 60/51 60/54 69/69 94/94 85/82 100/99 99/99 89/88 100/100 84/77 51/<50 100/100 94/92 100/98 92/87 100/100 83/72 98/98 100/99 95/95 75/<50 90/95 100/100 Figure 1. Maximum likelihood (ML) bootstrap 50% majority-rule consensus tree based on ITS data for Andeimalva and its Malvoideae relatives. ML/parsimony bootstrap values are indicated above branches. A new large-flowered species of Andeimalva... 95 Taxonomy Andeimalva peruviana Dorr & C.Romero, sp. nov. urn:lsid:ipni.org:names:77191589-1 Figure 2 Type. PERU. La Libertad: Bolivar, alpine bogs around Rio Negro and Pampa Uchulala along road to Bambamarca, 07°13'51"S, 077°38'21"W, 3750 m, 3 June 2015 (fl), C. Vega Ocaña 419 with R.W. Bussmann, N. Paniagua Zambrana, F. Díaz Llajo & F. Díaz Vega (holotype: MO-2423556!; isotypes: HAO, US-01184179!, USM). Diagnosis. Differs from Andeimalva mandonii (Baker f.) Kearney in having larger stipules (13–15 × 5–8 mm versus 5–12 × 1–2 mm) that are broadly subulate (not filiform), and larger calyx lobes (1.7–2.1 × 1–1.8 cm versus 0.5–1 × 0.3–0.5 cm) and petals (3.5–4.5 × 2.5–3.2 cm versus 1.5–3 × 1.5–2 cm). Description. Shrubs, 1.5–3 m tall; young stems densely lanate and appearing white, glabrescent and dark brown to almost black in age. Stipules subulate with broader cor- date base, 13–15 mm long, 5–8 mm wide, lanate with appressed stellate hairs, venation with 3 prominent parallel primaries running their length and up to 8 at base, persistent beyond life of leaf. Leaves simple, spirally arranged, petiolate, petioles 4–6 mm long, lanate; blades lanceolate to narrowly lanceolate, 5–8.2 cm long, 0.8–1.8 cm wide (lower leaf measurements due to serial size reduction toward the apex of flowering branches), base cordate, slightly asymmetric, apex acute, discolorous, drying dark green to brown above and whitish-tan below, bullate above with scattered sessile stellate hairs of vary- ing sizes, the arms on the larger hairs ascending, midrib above densely pubescent and appearing white, densely lanate below with sessile stellate hairs and larger stalked, multi- rayed stellate hairs, the arms on the larger hairs spreading, stalks dark-colored, margin crenate-serrate, venation pinnate with 13–18 secondaries per side. Flowers solitary or in 2–3(–4)-flowered axillary or pseudo-terminal cymes, ca. 5–7 cm in diameter at anthesis, pedicellate, pedicels to 0.5 cm long; involucral bracts (2–)3, 1–1.8 cm long, 0.5–0.8 cm wide, inserted just below calyx, densely stellate pubescent, hairs near margin long- stalked and multi-rayed. Calyx lobes broadly deltoid, acuminate, slightly unequal in size, 1.7–2.1 cm long, 1–1.8 cm wide, pubescent within especially toward base, stellate- pubescent on the outside with small and large multi-rayed hairs. Petals obovate, un- equal in size within the flower, 3.5–4.5 cm long, 2.5–3.2 cm wide, slightly to markedly asymmetric, deep mauve or dark magenta-purple, apex slightly undulate, unguiculate, glabrous except appressed simple hairs near base within and claw margins densely pubes- cent. Staminal column 2.8–3.2 cm long, sparingly pubescent throughout with simple hairs; anthers numerous (100+), 0.7–1.4 mm long, clustered at upper half of column; free portion of filaments (1–)1.6–5(–6) mm long. Carpels 10, cells uniovulate. Stigmas 20, capitate, scarcely exceeding the anthers at anthesis. Mericarps and seeds unknown. Distribution and ecology. At present, known only from the type locality where it occurs in alpine bogs at 3750 m. On the label of the type specimen there is a slight discrepancy regarding the political subdivision for the locality. Google Earth Pro mapping (8 June 2011 imagery, https:// Laurence J. Dorr et al. / PhytoKeys 110: 91–99 (2018)96 Figure 2. Illustration of Andeimalva peruviana. A Habit B detail of leaf below C detail of leaf above: note bullate surface D stalked and sessile stellate trichomes from leaf blade below E involucral bracts subtending ca- lyx (only 3 calyx lobes visible) F petal G staminal column: note stigmas scarcely exserted at apex H style with 20 capitate stigmas I detail showing two stigmas J detail showing two anthers (Source: Vega Ocaña 419, US). A new large-flowered species of Andeimalva... 97 www.google.com/earth/) places the coordinates cited on the label (presumably derived from a GPS device and for a logical locality near a road crossing of a wet drainage) in San Martín, and just over the eastern border delineated for La Libertad. However, we cannot verify how well Google Earth Pro finely draws political boundaries in relation to those that might appear on official government maps of such remote regions. Etymology. The specific epithet is derived from the name of the country (Peru) where the new species is found. Preliminary conservation status. Following the criteria and categories of IUCN (2017), Andeimalva peruviana is given a preliminary status of Vulnerable (VU D2) due to population very small or restricted (area of occupancy < 20km2 and number of lo- cations < 5). While the type of locality is quite remote and unexplored habitat occurs nearby, the general area has no protected regions and might be subject to habitat degra- dation from grazing or changes in hydrology. The latter includes human modification to its drainage system or climate change affecting montane precipitation. Discussion Morphologically Andeimalva peruviana appears to be closer to the three anomalous species of Tarasa first united by Krapovickas (1965) and later transferred to Andeimalva by Tate (2003), which share leaf morphology and mericarps with apical awns. Andei- malva chilensis, sister to the crown group (Fig. 1), has greater morphological diver- gence. However, its lack of awns relative to the other species of Andeimalva (uncertain in A. peruviana) may be an autapomorphic loss given that related genera such as Tarasa mostly possess awns (see Tate 2003). The chromosome number of our new species is unknown, but it would prove informative for generic affiliation. Optimal trees place A. peruviana as sister to A. mandonii, which has ML but not parsimony bootstrap support. This affinity is reflected in the morphology with shared generally larger floral features relative to the other species, but notably differs in stipule morphology (see Key below and Diagnosis). The large stipules of A. peruviana with prominent parallel major veins differ from those of the other species that are narrow in width and attachment. Most species of Andeimalva are geographically well separated. However, A. spici- formis (Krapov.) J.A. Tate and A. peruviana are nearly sympatric on a gross scale with both occurring at high elevation in La Libertad, Peru. Nonetheless, the two species appear to be separated by altitude, hydrology, and a physical barrier. Andeimalva spici- formis with its scattered distribution in northwestern Peru at 2400–3200 m, is known in La Libertad from a single collection (A. López M. 1439, US; see Tate 2003: 15) made at 2600 m. In contrast, the type locality of A. peruviana, about 70 km distant near the border of La Libertad, was made at 3750 m and is physically well separated by an east-west divide created by the deep valley of the Rio Marañon. The boggy habitat of A. peruviana in a region rich in small alpine lakes near the wet eastern side of the An- des generally appears much wetter than where A. spiciformis grows on drier mountain areas to the west where the Andean rainfall shadow has greater influence. Laurence J. Dorr et al. / PhytoKeys 110: 91–99 (2018)98 Key to Species of Andeimalva (modified from Tate 2003) 1 Leaves suborbicular, slightly 3–5-lobed; mericarps without an apical awn; central and southern Chile .......................................................... A. chilensis – Leaves lanceolate, not lobed; mericarps with prominent apical awn (or char- acter state unknown); Bolivia, Peru .............................................................2 2 Flowers in dense axillary glomerules; petals notched apically; mericarp awns 1–1.5 mm long; Peru ..............................................................A. spiciformis – Flowers solitary or in few-flowered axillary cymes; petals not notched api- cally; mericarp awns 1.5–4 mm long (or character state unknown) .............3 3 Staminal column < 2 mm long; calyx lobes 0.4–0.5 cm long; petals < 2 cm long; Peru ......................................................................A. machupicchensis – Staminal column > 2 mm long; calyx lobes 0.5–2.1 cm long; petals 1.5–4.5 cm long ......................................................................................................4 4 Petioles 3–12(–20) mm long; stipules 5–12 × 1–2 mm, filiform; staminal column 0.3–0.8 cm long; calyx lobes 0.5–1 cm long; petals 1.5– 3 cm long; Bolivia ......................................................................................A. mandonii – Petioles 4–6 mm long; stipules 13–15 × 5–8 mm, broadly subulate; staminal column 2.8– 3.2 cm long; calyx lobes 1.7–2.1 cm long; petals 3.5–4.5 cm long; Peru ................................................................................A. peruviana Acknowledgments We thank Alice R. Tangerini (US) for her botanical illustration and James C. Solo- mon (MO) for arranging the loan of type specimens to US. The laboratory work was conducted in, and with support from, the Smithsonian’s Laboratories of Analytical Biology of the National Museum of Natural History. Finally, we would like to thank Carlos Vega, Rainer Bussmann, Narel Paniagua, Francisco Díaz-Llajo, and Francisco Díaz-Vega, the collectors of the type specimens. Reviewers Margaret Hanes, Mersedeh Pejhanmehr, Jennifer Tate, and anonymous made helpful suggestions for improving an earlier version of this manuscript. References Bates DM (1963) The genus Malacothamnus. PhD Thesis, University of California, Los Angeles. Bates DM, Blanchard Jr OJ (1970) Chromosome numbers in the Malvales. II. New or other- wise noteworthy counts relevant to classification in the Malvaceae, tribe Malveae. 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