﻿Maxillariaanacatalinaportillae (Orchidaceae, Maxillariinae), a new remarkable species from Ecuador

﻿Abstract Neotropical genus Maxillaria Ruiz & Pav. belongs to one of the most diverse and species-rich groups of orchids. Several of its representatives are popular, horticultural plants with large and showy flowers, often nicely fragranced. It is not uncommon that some distinctly colored individuals are introduced to the commercial market under names of similar, more or less related species, as informal varieties or color forms, largely causing confusion. While investigating the diversity of Maxillaria in Ecuador, we have encountered plants that were commercially referred to as M.sanderianaxanthina. In the course of conducted morphological and micromorphological analyses, we concluded that it is a new, separate species and hereby, we describe it as M.anacatalinaportillae.


Introduction
Maxillaria Ruiz & Pav. is one of the most interesting species groups in the orchid family. For many years it has been, and in some way still is quite a controversial genus. The lack of clearly defined boundaries of Maxillaria sensu stricto resulted in proposing several taxonomic approaches of the subtribe Maxillariinae Benth. over the past few decades. For a long time, it has been suspected that it is an assemblage of taxa, consisting of morphologically disparate groups of species (Whitten et al. 2007). Establishing the exact number of species belonging to the various genera or the subtribe itself is not easy since it depends mainly on the adopted classification system and genus concept. Maxillaria covers about 4/5 of species belonging to the subtribe (Senghas 2002). Depending on the applied classification, it counts from approximately 420 (Dressler 1993), through 634 (Schuiteman and Chase 2015) to 750 species (Senghas 2002).
One of the most spectacular groups of species within the genus is often referred to as 'Maxillaria grandiflora-complex' or also alliance/group (Dodson 1997), it includes species such as M. platypetala Ruiz & Pav., M. molitor Rchb. f., or M. sanderiana Rchb. f. ex Sander. Their common feature is the size of flowers -generally large and showy, sometimes also brightly colored. The main reason for this reference is the supposed similarity to Maxillaria grandiflora (Kunth) Lindl. (Christenson 2013). Dodson (1997) characterized this group as caespitose plants with unifoliate pseudobulbs and foliaceous leaf sheaths, petiolate leaf, large flowers (5-12 cm in diameter), and broad and blunt sepals and petals. Christenson (2013) pointed out, however, that the type specimen of M. grandiflora itself is bifoliate and several other species included by Dodson, do not fulfill the morphological criteria presented above (e.g. M. napoensis Dodson, M. batemanii Poepp. & Endl). Indeed, for many years there have been some ambiguities about the type of M. grandiflora (Fig. 1) and its locality, however, they have been clarified by Blanco and Stauffer in 2011. Detailed analysis of the collection time, numbers and travel route conducted by them, have led to a conclusion that the type locality of Maxillaria grandiflora must be somewhere in the eastern part of La Cruz municipality in the department of Nariño, Colombia at the altitude of 2,067 m. As mentioned above, some questions were raised by Christenson (2002aChristenson ( , b, 2013 about the number of leaves on the pseudobulb of the type specimen. It is essential to clarify that the type collection of Maxillaria grandiflora consists of three sheets deposited in the Bonpland herbarium in the Muséum National d'Histoire Naturelle (P) in Paris. In their paper, Blanco and Stauffer (2011) state clearly that in this case we are dealing with a mixed collection. During our visits in P herbarium, we have examined this specimen and we agree with Blanco and Stauffer that it consist of plant parts that belong to at least two different species. As the number of apical leaves per pseudobulb is an important taxonomic feature, the existence of a pseudobulb with two apical leaves as part of the type collection of M. grandiflora might have been a source of confusion resulting in Christenson`s belief that this taxon is not a member of the M. platypetala Ruiz and Pav. alliance (sensu Whitten et al. 2007). Christenson (2013) pointed out three useful clues for the proper taxonomic identification within Maxillaria grandiflora-complex: 1) the orientation of tepals is crucial for understanding the species circumscription (segments reflexed, inflexed, incurved, among others), 2) flower color and lip markings, 3) the shape of the lip apex (transversely terminated, usually undulate, with lobes similar to the ruffles of a petticoat or rigidly held and V-shaped in cross-section). Most of the large-flowered Andean Maxillaria species are at times informally referred to as Maxillaria grandifloracomplex. This often causes confusion as many of them have little or nothing in common with actual M. grandiflora (Kunth) Lindl. The general understanding of these species remains scarce and requires intensified taxonomic investigation. Especially three   Christenson (2013) some may consider them to be two ends of a spectrum of variation over a broad geographic range and in this concept, the name M. grandis takes priority and is the correct name for the broadly defined species. The variability within M. sanderiana may also suggest that, in fact, it is a complex of species consisting of one or more undescribed taxa. Another theory has been mentioned by Blanco as a comment in Christenson`s monograph (2013). He suggested that M. wojii is simply a color variation of M. grandis or that both are hybrids of M. sanderiana with some other species in the Maxillaria grandiflora-complex. This would expend the occurrence range of M. grandis as M. wojii is known only from Colombia.
During the past few years, we have been working intensely on the classification and especially species delimitations within Maxillaria sensu stricto. We have investigated herbarium materials deposited in most of the world's collections, such as AMES, B, C, COL, MA, MO, P, W, W-R to name just a few. During our expeditions to South America, our attention was drawn to the Maxillaria grandiflora-complex and many taxonomical ambiguities it brings. We have collected samples from both commercial and hobby growers but also purchased several plants to cultivate them in the greenhouses of the University of Gdańsk. One of them was identified as M. sanderiana and was referred to by Ecuagenera as "xanthina". Indeed, according to Christenson (2013), there are two color forms of M. sanderiana: atropurpurea (H. Williams) Christenson and xanthina Christenson. However, after morphological analysis of the flower, it became clear that we are facing new species and hereby we describe it as a new one. The new entity can be easily distinguished by having flat margins of petals, which are gently angled, callus extending beyond the middle of the lip and form of lip middle lobe, which is broadly cordate or triangular when spread, with fold down margins. It is known from several plants that are available commercially, however, they all originate from single (type) population.

Morphological analysis
Flowering plants of the new species were collected on November 11 th , 2003 in the Carchi province (northern Ecuador). The species was photographed in situ and taken to cultivation in the greenhouses of Ecuagenera Cia. Ltda with initial identification as M. sanderiana 'xanthina'. In 2020, Ecuagenera provided plant material consisting of five plants which have been sent to Poland with corresponding CITES certificates.
Plants have been cultivated in the greenhouses of the University of Gdańsk (voucher 0148255) and used for the presented analysis. Herbarium specimen were prepared to be used as type material and deposited at UGDA. Particular parts of the flower were dissected, measured, and drawn under stereomicroscope. The line illustration of the new species was prepared from material preserved in Kew Mixture (53% ethanol: 5% formaldehyde: 5% glycerol: 37% water) and digital photos. The new entity has been compared with more than 800 herbarium specimens of other members of Maxillaria grandiflora-complex from the following herbaria: AMES, B, C, COL, MA, MO, NY, P, W, W-R, VALLE, QCE, and QCNE. We conducted a careful comparison of the new species with the protologues and type material of all species belonging to the complex, as well as regional floras and checklists such as Dodson and Marmol (1980), Dodson (2002), and Jørgensen and León-Yánez (1999). The conservation status of the new species was evaluated, based on the guidelines of the International Union for Conservation of Nature (IUCN 2019).

Phylogenetic analysis
Plant material for molecular analysis has been obtained from plants provided by Ecuagenera and living orchid collection of the University of Gdańsk. Remaining sequences were obtained from NCBI database. The GenBank accession numbers of the used sequences in the study are given in the Appendix 1: Table A1.
Molecular substitution model was based on AIC (Akaike information criterion) and calculated with PhyML website (http://www.atgc-montpellier.fr). The GTR+G+I model was selected as the best one for studied matrix. In the first step of phylogenetic tree reconstruction, two separate matrices (for ITS and matK) were analyzed using Bayesian Interference and maximum likelihood methods. Finally, due to the low clade support, high polytomy, and no sign of topology conflict, the combined analysis was performed (only the results of this one are shown, Fig. 2). The results of single markers analysis are available upon request.
The Bayesian analyses was performed using Markov chain Monte Carlo (MCMC) in MrBayes 3.2.7a (Huelsenbeck and Ronquist 2001) using CIPRES Science Gateway (Miller et al. 2010). The analysis was performed in two simultaneous runs of four chains for 20,000,000 generations, sampling one tree for each 100, until the average standard deviation of split ranges reached a value < 0.01. TreeAnnotator v. 1.8.1 (Drummond et al. 2012) were used to construct a maximum clade credibility tree, with a burn-in of 25%. The support of the clades was evaluated by the posterior probability (PP).

Micromorphological analysis
Samples for the scanning electron microscopy (SEM; voucher no. UGDA.0148255) have been preserved in 2,5% GA and 2,5% PFA in 0,05M cacodylate buffer (pH 7,0). Following dehydration in an ethanol series, they were dried by the critical point method using liquid CO2 and coated with gold. They were observed using a Philips XL-30 scanning electron microscope.  Description. Plants caespitose. Pseudobulbs 4-5.5 cm long, 4-4.5 cm wide, ellipsoid to almost orbicular, laterally compressed, unifoliate, supported basally by 1-2 leafy sheaths. Sheaths petiolate; petiole up to 20 cm long, conduplicate, narrow; blade up to 30 cm long and 8.5 cm wide, ligulate to oblong-elliptic, acute to shortly acuminate at apex, basally cuneate. Leaf petiolate; petiole up to 5 cm long, conduplicate; blade up to 33 cm long and 7.5 cm wide, similar in form to sheaths, ligulate to oblong-elliptic, acute to shortly acuminate. Peduncle ca 5-7 cm long, enveloped in 4-5 sheaths, erect, basal, single-flowered; sheaths elliptic-lanceolate, acute, thin, fibrous, brownish. Flowers large and showy, scentless, campanulate, not fully opened, resupinate, sepals red-wine or maroon outside, yellow inside with redmaroon basal part (Fig. 3); petals yellow with red-maroon veins and irregular dots on both sides of the middle vein, lip basal part yellow, callus yellow with dark apical part, middle lobe red-black with grayish suffusion, margins yellow, red-maroon outside, gynostemium yellowish with red-maroon on the ventral surface below stigma, anther yellow. Floral bracts ca 60 mm long, elliptic-lanceolate, greenish-brown with maroon veins. Ovary 30 mm long, glabrous. Tepals thick, fibrous. Dorsal sepal 60-62 mm long, 25-27 mm wide, elliptic-ovate, concave along midvein, apex subobtuse, canaliculated. Petals 60-62 mm long, 23-25 mm wide, oblong-lanceolate to ligulatelanceolate, falcate at base, apex attenuate, thickened, subobtuse. Lateral sepals 75 mm long, 30-32 mm wide, obliquely oblong triangular, somewhat concave at the base, apex thickened, subobtuse. Lip hinged on the column foot, ca 45 mm long in total, 30 mm wide when spread, very stiff, gently arched, papillate in the apical half, 3-lobed in the apical third, callus very massive reaching beyond the midpoint of the lip, ligulate-ovate, flanked by narrow wing on each side; middle lobe ca 13-15 mm long, 18-20 mm wide, broadly cordate or triangular when spread, concave along midvein, margins crenulate-undulate, fold-down; lateral lobes 30 mm long, oblongovate in outline, canaliculated in natural position. SEM analysis revealed the presence of copious moniliform trichomes and pseudopollen grains on the lip surface, mainly middle lobe and callus (Fig. 5). Lip base and lateral lobes were rather smooth, with villiform to obpyriform papillae towards the middle part of the lip (Fig. 5B). These papillae seem to be the early stage in the development of the moniliform trichomes. Gynostemium 23 mm long, column foot 33 mm long, apically upcurved, clinandrium densely glandular.

Maxillaria anacatalinaportillae
Ecology and distribution. Grows as an epiphyte in cloud rainforest at altitude of 1700 m asl, known only from the type location.
Eponymy. Name dedicated to Ana Catalina Portilla Schröder -daughter of Alex Portilla, finder of the new entity.
Conservation status. According to the IUCN Red List criteria (IUCN 2019), the new entity should be classified as critically endangered (CR B2ab (iii)), based on the small number of known populations and restricted area of distribution. The province of Carchi in recent years suffered from problems caused by climate change, anthropogenic impact on the environment, and the lack of awareness of natural resources. The change of land use, expansion of the agricultural frontier, population growth, or the opening of new roads are some dynamics that generate pressure on the ecosystems, compromising the ecological processes that take place in them. According to Global Forest Watch, from 2001 to 2020, Carchi lost 8.34 kha of tree cover, equivalent to a 3.7% decrease in tree cover since 2000.

Notes.
We know about several living collections in Ecuador that are probably representing the new entity, however, since we were not able to investigate these plants in person, we can only treat them as possible representative specimens. According to A. Hirtz, collections are located in Botanic Garden of Quito, Orquidario Las Juntas (near Gualtal at the south side of the Golondrinas Volcano, owned by Arturo and Esmeralda Mendez), Quinche (near the airport of Quito, collection of Juan Galarza), and Orquidario Casa Dracula in Quinshul (owned by Hector Yela).
Phylogeny. The results are presented on the maximum clade credibility tree obtained from Bayesian analysis. Support of particular clades (PP and additionally BS -from ML analysis) is marked with a square circle or pentagon, according to the legend given on the Fig. 2. Obtained phylogenetic tree consists of representatives of Maxillariinae, including those recently recognized by some authors (e.g. Szlachetko et al. 2012) genera Calawaya (III) and Pseudocymbidium (represented by M. lueri Dodson = Pseudocymbidium lueri (Dodson) Szlach. and Sitko). Maxillaria s. str. (II) seems to be monophyletic, but there is no PP or BS support for this clade. The clade of Maxillaria grandiflora-complex is well supported and includes the new species Maxillatia anacatalinaportillae.

Phylogeny
The main purpose of phylogenetic reconstruction in this paper was the placement of the new species, thus the phylogenetic relationship within the Maxillarinae will not be widely discussed here. Our results indicate the affinity of Maxillaria anacatalinaportillae to the Maxillaria grandiflora-complex. However, the relationships between species within the complex are still unclear. Moreover, some authors (e.g. Whitten et al. 2007) postulated to include the complex into Maxillaria platypetala alliance, due to the relatively low genetic differentiation among the members of these two groups, which may suggest the recent radiation of the species. In our opinion further researches are necessary to make any decision about the taxonomic position of mentioned taxa and to fully resolve the relationships between its species.

Morphology
Many representatives of Maxillaria grandiflora-complex (sensu Christenson 2013) or M. platypetala alliance (sensu Whitten et al. 2007) are superficially similar to each other but can be easily distinguished when live plants are compared side by side (Blanco and Stauffer 2011 and references their). Some of the distinguishing features, such as color patterns, may disappear or be obscured in dried herbarium specimens; especially flowers tend to become dark brown to almost black when dry, regardless of their original color (Blanco and Stauffer 2011).
Maxillaria anacatalinaportillae is the only species morphologically similar to M. sanderiana (Fig. 6), M. grandis (Fig. 7), and M. wojii having flowers with yellow as basic color. Flowers of all aforementioned species are primarily white with various degrees of red-wine or maroon saturation.
The forms of the flower of Maxillaria sanderiana and M. anacatalinaportillae are quite similar, but, again, the lip middle lobe of the former has strongly undulated and planar margins, and petals are shorter than dorsal sepal (vs equal in length in our new species). The micromorphology seems to be the same -in both cases, the lip surface is predominantly covered with pseudopollen, which is formed by the fragmentation of multicellular, moniliform trichomes (Davies et al. 2000). The populations of  Table 1.  Figure 7. Drawing of the flower parts of Maxillaria grandis A lateral sepal B petal C dorsal sepal D lip. Scale bars: 10 mm. Redrawn from the type by N. Olędrzyńska.

Micromorphology
It is estimated that as many as 56% of the representatives of Maxillaria sensu lato attract pollinators with "empty promises", which are a combination of visual, tactile, and olfactory stimuli (Davies et al. 2005 and references therein). Among the species that offer any kind of reward, there are three types: nectar, pseudopollen (farina), and waxlike substances (Davies et al. 2003a(Davies et al. , b, 2005. Until now, the micromorphology of only twelve members of Maxillaria grandiflora-complex has been studied. A common feature among its representatives is the presence of pseudopollen and at least 30 Ecuadorian species of Maxillaria produce pseudopollen (Davies et al. 2000). M. anacatalinaportillae is no exception and SEM analysis revealed the presence of copious moniliform trichomes and pseudopollen grains on the lip surface, mainly middle lobe and callus. Lip base and lateral lobes were rather smooth, with villiform to obpyriform papillae towards the middle part of the lip. (Davies and Turner 2004). Pseudopollen is usually produced by the fragmentation of labellar trichomes and it has the form of a whitish layer with a powder-like structure. It can be considered as a substitute reward for real pollen. The presence of obpyriform and moniliform trichomes is typical for members of the Maxillaria grandiflora-complex (Davies and Turner 2004;Lipińska and Kowalkowska 2018) and is not surprising, since wherever pseudopollen-forming trichomes occur, labellar papillae tend to be obpyriform (Davies and Turner 2004).
The main pollinators of Maxillaria are stingless bees (Meliponini) (Roubik 2000). According to Davies et al. (2000), some of the members of the Maxillaria grandifloracomplex are pollinated by different insects: Maxillaria fletcheriana Rolfe by the bumblebee Bombus volucelloides Gribodo, M. grandiflora and M. sanderiana by Eulaema cingulata Fabricius. It is believed that bees collect pseudopollen from the flowers because of the nutrients it contains (Davies et al. 2000) and these include starch, oils, and proteins (van der Pijl and Dodson 1966). This may suggest that M. anacatalinaportillae is also pollinated by bees, similarly to closely related M. grandiflora and M. sanderiana.