Introduction
Thegenus BlitumL. [tribe Anserineae Dumort., Chenopodiaceae Vent. sensuHernández-Ledesma et al. 2015; some authors, e.g. APG IV (2016) consider Chenopodiaceae as included in Amaranthaceae Juss.], traditionally included in Chenopodium L. s.l. during the last centuries (with a few exceptions), was recently taxonomically resurrected by Fuentes-Bazan et al. (2012a, b) based on phylogenetic studies of Chenopodioideae Burnett, where a reclassification of the whole subfamily was proposed recognizing four tribes and ten genera. The generic classification outlined in Fuentes Bazan et al. (2012b) has been updated in many recent publications (e.g., Iamonico 2012, Mosyakin 2013, Uotila 2011, 2017, Sukhorukov et al. 2013, Sukhorukov and Kushunina 2014) and generally accepted (e.g., Hernández-Ledesma et al. 2015; some online floras and databases, e.g., French flora; POWO 2021-onward).
Blitum (incl. Agathophytum Moq., Monolepis Schrad., Scleroblitum Ulbr., Carocarpidium S.C.Sand. & G.L.Chu) is currently known to include about 12 species and nothospecies (see Fuentes-Bazan et al. 2012b, Mosyakin 2013, Hernández-Ledesma et al. 2015, Sukhorukov et al. 2018), ten of which occur in the Northern Hemisphere [B. asiaticum (Fisch. & C.A.Mey) S.Fuentes, Uotila & Borsch (= Monolepis asiaticaFisch. & C.A.Mey.), B.bonus-henricus (L.) Rchb., B. californicum S.Watson, B. capitatum L. s.l., B. korshinskyi Litv., B. litwinowii (Paulsen) S.Fuentes, Uotila & Borsch, B. nuttalianum Schult. (= Monolepis nuttalliana (Schult.) Greene), B. petiolare Link (= Chenopodium exsuccum (C.Loscos) Uotila), B. ×tkalcsicsii (H.Melzer) Mosyakin, B. virgatumL. (= Chenopodiumfoliosum Asch.)], one in South America (B. antarcticum Hook. f.) and one [B. atriplicinum F.Muell. (= Scleroblitum atriplicinum (F.Muell.) Ulbr.)] in Australia.
As part of the ongoing study on Chenopodium s.l. (e.g., Iamonico 2010, 2011, 2012, 2013, 2014, Iamonico and Jarvis 2012, Mosyakin and Iamonico 2017), a population belonging to the genus Blitum was found in the Dolomites Massif (North-Eastern Italy). After a detailed analysis of pertinent literature, examination of specimens in herbaria and in virtual herbaria online (type material included), and molecular investigation, we reached the conclusion that the population found cannot be identified as belonging to any known speciesof Blitum. Consequently, we describe here a species new for science and compare it with the related taxa.
Material and methods
Morphological and nomenclatural study
The work is based on field surveys, analysis of relevant literature (protologues of the names involved in the present study are included), and examination of specimens preserved in the following herbaria: BR, GH, K, LINN, NY, P, RO, SD, UC, and US (codes according to Thiers 2022). Photographs of inflorescence and fruits were made using an optical stereoscope Wild Heerbrugg M8.
Molecular study
Genomic DNA was extracted using the Genomic DNA purification kit (Thermo Scientific™, Waltham, MA, USA). The nuclear ITS region and the plastid trnL-F region were amplified following Fuentes-Bazan et al. (2012a). The obtained PCR products were cleaned using the HT ExoSAP-IT (Applied Biosystems™, Waltham, MA, USA) and sequencing was carried out at the Eurofins Genomics Sequencing Service (Germany), with the same primers employed in the amplification reactions. For sequencing of the trnL-F locus, two internal primers were also employed as reported in Fuentes-Bazan et al. (2012a). The GeneStudio sequence analysis software (http://genestudio.com/) was used to assemble the final consensus sequences. The new sequences were deposited in the International Nucleotide Sequence Database Collaboration (INSDC) repositories, through the EuropeanNucleotide Archive (ENA) platform, with the following GenBank accession numbers: OU753550 (ITS region) and OU753551 (trnL-F region).
The obtained sequences were compared with those present in the INSDC archives using the BLAST program available at the USA National Center for Biotechnology Information (NCBI) web server (http://www.ncbi.nlm.nih.gov). Two distinct datasets of ITS and trnL-F sequences were created, including the newly obtained sequences and other suitable sequences available in the INSDC repositories, following the most recent classifications for the genus Blitum (Fuentes-Bazan et al. 2012b, Sukhorukov et al. 2018). For each analysis, a sequence of Chenopodium pallidicaule Aellen (INSDC accession number: HE577438 for the ITS locus; INSDC accession number: HE577573 for the trnL-F locus) was used as outgroup to orient the tree. Multiple sequence alignments were generated with MUSCLE (Edgar 2004) and used for phylogenetic analyses. The ITS multialignment included 15 sequences for a total of 507 aligned positions and the trnL-F multialignment included 13 sequences for a total of 987 aligned positions.
Phylogenetic analyses were performed with MEGA v. X program (Kumar et al. 2018) using the Maximum Parsimony (MP) and the Maximum Likelihood (ML) methods. For ML, the model that best fit the data, found with the “Find best DNA Models” tool implemented in MEGA v. X under the BIC criterion (Schwarz 1978), was T92 + G for both the molecular markers. Nonparametric bootstrap re-sampling (Felsenstein 1985) was performed to test the robustness of the obtained topologies (1000 replicates). Bayesian Inference (BI) analyses were carried out with MrBayes version 3.1.2 (Ronquist and Huelsenbeck 2003). The analyses included two independent MCMC runs, each composed of four chains (three heated and one cold); each MCMC ran for 1 × 106 generations, sampling trees every 100 generations. The sampling of the posterior distribution was considered to be adequate if the average standard deviation of split frequencies was ≤ 0.01. The first 2500 trees were discarded as burn in, as determined by stationarity of log likelihood assessed using Tracer version 1.5 (Rambaut and Drummond 2007). The consensus topology and posterior probability values were then calculated from the remaining trees. The final tree pictures were created with Inkscape v. 0.92 (Figs. 1a, 1b).
Alignments of the ITS and trnL-F sequences of different Blitum species were also obtained with MUSCLE (Edgar 2004) to calculate the percent identities within the genus (Figs. 1c, 1d).
Results
Morphology
The genus Blitum is morphologically characterized by having stems often unbranched or nearly so, or mainly branched at or near the base, leaves often forming basal rosettes, stigma lobes 2–4, and seed vertical with the seed coat displaying a visible protoplast (Fuentes-Bazan et al. 2012b, Iamonico 2013, Sukhorukov et al. 2018). The genus is rather heterogeneous morphologically (Clemants and Mosyakin 2003, Zhu et al. 2003, Sukhorukov et al. 2018), the plants we found in the Dolomites Massif (North-Eastern Italy, Veneto region) appear to resemble both B.bonus-henricus L. and B. californicum S.Watson. In particular, they show the following combination of characters: perennial (other Blitum species, exceptperennial B. bonus-henricus and B. californicum, are usually annual), erect to ascending [B.asiaticum (Fisch. & C.A.Mey.) S.Fuentes, Uotila & Borsch, B. litwinowii (Paulsen) S.Fuentes, Uotila & Borsch, and B. nuttalianum Schult. have stems prostrate], basal rosette with many leaves [B.asiaticum and B.nuttalianum have just 1 or 2 leaves, that may wither away completely by anthesis], large and more or less triangular leaves with their base hastate or cordate, more than 5–6 cm long [the other species have leaves more or less rhomboid, with base cuneate, and blades usually up to 5–6 cm long (B. virgatum L. and B. capitatum L. have leaves triangular and up to 8 cm long, but they are annual species)], perianth with 5 segments (other species of the genus usually have 1–3 segments) not becoming red and succulent at the fruiting stage (in B.virgatum and B.capitatum perianth segments are fleshy and red at maturity, forming a berry-like structure).
In comparison with Blitum bonus-henricus (native in Europe, introduced in North America) and B.californicum [endemic to California and Mexico (Baja California)], the Italian population differs morphologically by both vegetative and generative characters, i.e. size, pubescence and leaf margins, colour of perianths segments, structure of the inflorescence, and size, colour, and surface of seeds (Tab. 1; see also the diagnosis under the taxonomic treatment).
Molecular data
In both phylogenetic reconstructions, those based on theITS and trnL-F markers (Figs. 1a, 1b), the sequences obtained from the type specimen collected in the Belluno Dolomites (Italy) resulted clearly separated from the other species of Blitum. In the ITS tree (Fig. 1a) our sequence was included in a well-supported clade (100/100/1.00) with a sequence ofB.bonus-henricus from Austria (HE577372). This phylogenetic relationship was not confirmed by the phylogenetic analysis based on the plastid spacer trnL-F (Fig. 1b), where the Belluno specimen and B. bonus-henricus were placed again as sister taxa, but without any statistical support.
On the basis of the percent identity matrices obtained with MUSCLE (Figs. 1c, 1d), the nucleotide divergences calculated between the Blitum sp. specimen from Belluno and each of the other recognized Blitum species ranged from 0.40% (Blitum sp. vs. B. bonus-henricus) to 4.37% (Blitum sp. vs. B. californicum) for the nucleotide ITS marker and from 0.86% (Blitum sp. vs. B. bonus-henricus) to 3.70% (Blitum sp. vs. B. petiolare) for the plastid trnL-F marker.
The interspecific divergences calculated between the ITS sequences of the currently taxonomically accepted species of this genus ranged from 0.20% [Blitumnuttalianum vs. B. asiaticum; B. capitatum subsp. capitatum vs. B. capitatum subsp. hastatum (Rydb.) Mosyakin; B. capitatum vs. B. litwinowii; B. capitatum vs. B. virgatum subsp. montanum (Uotila) S.Fuentes, Uotila & Borsch] to 3.98% (B.californicum vs. B. petiolare; B. californicum vs. B. antarcticum). The ITS sequence of B. litwinowii and that of the subspecies B. virgatum subsp. montanum did not show divergences. For the plastid trnL-F spacer, the interspecific divergences ranged from 0.21% (B. litwinowii vs. B. virgatum subsp. virgatum) to 4.47% (B. bonus-henricus vs. B. petiolare).
Therefore, for both analysed markers, the nucleotide divergences calculated between the sequence of Blitum sp. from Belluno and other Blitumspecies are comparable with the interspecific divergences calculated inside this genus.
Discussion
The results obtained in the present study, both morphological and molecular data, support the description of the population from the Belluno Dolomites as a new species, Blitum venetum, which is formally described below.
Blitum venetum can be reliably distinguished from the closely related species B. bonus-henricus and B. californicus by various vegetative and generative morphological characters. All diagnostic characters found for B. venetum have high taxonomic value in the classification of both Blitum and the intrageneric taxa of Chenopodium s.l. (Mosyakin and Clemants 1996, Clemants and Mosyakin 2003, Zhu et al. 2003, Sukhorukov et al. 2018). Its recognition as a distinct species is further supported by its phylogenetic position in both nuclear ITS and plastid trnL-F trees, as well as considering, for both of the analysed markers, the nucleotide divergences among various Blitum species.
Blitum venetum Iamonico, Argenti, Sciuto & Wolf, sp. nov.
Holotype. Italy, Veneto region, Belluno administrative Province, Vigo di Cadore Municipality, locality Casera Razzo, 46°28’41.8”N, 12°36’33.3”E, 1740 m a.s.l., nitrophilous grasslands, 11 August 2020, C. Argenti s.n.; holotype: RO! (Fig. 2).
Diagnosis (Fig. 3) – Blitum venetum differs from B. bonus henricus and B. californicum in having the leaves wider (11–13 cm vs. 3–10 cm and 3–9 cm, respectively), glabrous (vs. adaxially sparsely farinose), with dentate and not undulate margins (vs. entire and/or undulate margins), the perianth segments with red or reddish terminal parts (vs. green throughout), the spike-like inflorescences bracteate, each floral glomerule subtended by a linear bract (vs. spiciform inflorescences ebracteate or nearly so), and the seed smaller (about 1 mm in diameter vs. usually 1.5–2.0 mm). In comparison with B.bonus-henricus, B.venetum also differs by the colour and surface of the seeds (blackish and alveolatevs. red and smooth; also B.californicum has reddish seeds).
Molecular vouchers. OU753550 (ITS region) and OU753551 (trnL-F region).
Description. Stems erect to ascending, branched, 40– 80(–90) cm, glabrous; sparsely inflated hairs in the inflorescence part. Leaves green, non-aromatic, blades glabrous; petiole 1–10 cm; blade triangular, 10–13 × 11–13 cm (size decreasing along the stem upwards), base truncate to hastate or cordate; margins dentate (in larger leaves with 6–8 teeth, teeth acuminate, 1.0–2.5 cm long), apex acute to acuminate. Inflorescences: glomerules mostly densely arranged in terminal and lateral spiciform structures, each subtended by a glabrous leaf-like bract (0.5–3.0 × 2–5 cm); the uppermost bracts with attenuate bases (edges of the leaf base forming an angle of about 90°), with two basal teeth (triangular) or with entire margins (lanceolate); each glomerule subtended by a linear bract (1.0–2.5 mm long), with membranous hyaline borders; some lower glomerules solitary and sessile, subtendend by the upper cauline leaves; spikes 6–8 cm long, the terminal one longer than the lateral ones; glomerules about 2 mm in diameter. Flowers: perianth segments connate up to the 1/3 of the total length, lobes 4 or 5, ovate, about 0.5 × 1.0 mm, scarcely keeled throughout, apex rounded, glabrous, green with reddish apical part; stamens 4 or 5; stigmas 2. Achenesobovoid, 1.5–2.0 mm; pericarp adherent. Seeds vertical, obovoid, about 1 mm in diameter, margins rounded; seed coat blackish, alveolate.
Etymology. The specific epiteth is dedicated to the Italian region (Veneto) in which the new species occurs.
Proposed vernacular name. Blito del Veneto (Italian), Veneto’s Goosefoot (English).
Flowering time. August to September.
Distributionarea and habitat. Blitumvenetum is known only from locus classicus, i.e., locality Casera Razzo (Belluno administrative Province, Veneto region, North-Eastern Italy), where it grows on nitrophilous grasslands at 1740 m a.s.l. The locus classicus is part of the territory of the Dolomiti Bellunesi National Park (date of establishment: 1988) and of the UNESCO World Heritage Site “The Dolomites” (date of inscription: 2009; see http://whc.unesco.org/en/list/1237).
IUCN Conservation assessment. Only one population wasfound (locusclassicus) and it occupies a small area (less than 50 m2). On the basis of criterion B3 of IUCN (2019), Blitum venetum is here assessed as Critically Endangered (CE).
Additional specimens examined
Blitum bonus-henricus. Habitat in Europae ruderatis, Herbarium Linnaeus no. 313.1 (LINN, lectotype). Belgium: Bastogne, 1884, A. Verheggen s.n. (P01089431); Theux, 18 June 1959, J. Pelgrims753 (BR0000027710821V). France:Orcières, village Merlette, 1830 m a.s.l. 13 July 1980, G.Sag 986 (P00036524). Italy: Lazio region, Montagna d. Duchessa (Velino), June 1946, B. Anzalone 10739 (RO); Lazio region, Piani di Livata al M. Autore, 21 July 1966, B.Anzalone 10737 (RO); Lazio region, Monti della Laga (Amatrice – RI), Fosso di Selva Grande, 1350-1500 m a.s.l., 6 July 1997, B.Anzalone 10734 (RO!); Trentino-Alto Adige, San Martino di Castrozza, August 1958, B. Anzalone s.n. (RO). Netherlands: Epen, July 1954, W. Voss s.n. (BR0000026765815V). Slovenia, Jesenice, 1200 m a.s.l., 18 August 1976, G. Sag 986 (P00041309). Sweden: Uppland, Bondkyrka parish, about 1½ km southwest of Berthåga, 18 September 1956, C.G. Alm 2972 (US03654843). Switzerland: Wegkant to Davos Platz, July 1975, W. Verbeke s.n. (BR0000025468366V). U.S.A.: New York, Long Island, East Hampton, 10 June 1877, E. S. Miller 321 (US03652612).
Blitum californicum. Mexico: Baja California, Ensenada, Sierra San Pedro Martir. Colony under live oak, Los Encinos, 10 August 1969, R. Moran 16539 (UC1361814). U.S.A.: California, San Diego county, in rocks at base of cliff ½ mile a southeast of Eel Point, 28 January 1967, R. M. Beauchamp 134 (SD00017034); San Diego county, west side of San Felipe Wildlife Area, under shrubs and mesquite, 26 March 1997, S. Bell 248 (SD); San Buena Ventura, March 1861, W. H. Brewer 244 (GH00936570, syntype, image available athttps://plants.jstor.org/stable/10.5555/al.ap.specimen.gh00936570); Fort Tejon, 1857-1859, J. Xantus de Vesey 104 (GH00936572, syntype, image available athttps://plants.jstor.org/stable/10.5555/al.ap.specimen.gh00936572); s.d., J. C. Fremont s.n. (NY00008484, syntype, image available athttp://sweetgum.nybg.org/science/vh/specimen-details/?irn=217758); s.d., D. Douglas s.n. (K000898461, syntype, image available athttp://apps.kew.org/herbcat/getImage.do?imageBarcode=K000898461).