INTRODUCTION

The genus Pachyphytum (Crassulaceae) was proposed by Link, Klotzsch et Otto (Klotzsch, 1841) with P. bracteosum Klotzsch (Moran, 1989) as the type species. Moran (1968) recognized three sections based on morphological affinities: Pachyphytum, Diotostemon and Ixiocaulon. Later, J. Thiede (in Eggli, 2003) merged the species of sect. Ixiocaulon into sect. Pachyphytum, resulting in only two sections: Diotostemon and Pachyphytum. Genetic distance-based classification of the genus employing AFLP molecular markers suggested two groups inside Pachyphytum (GarcíaRuíz 2003). In this classification, group A included species P. bracteosum, P. brevifolium, P. glutinicaule, P. hookeri, P. longifolium, and P. oviferum; while group B incorporated P. caesium, P. coeruleum, P. compactum, P. contrerasii, P. fittkaui, P. garciae, P. kimnachii, P. machucae, P. rzedowskii, P. viride, and P. werdermannii. It should be noted that neither Moran´s nor Thiede´s groupings match GarcíaRuíz´s results.

A recent molecular phylogenetic analysis of the genus Echeveria and related genera including Pachyphytum, based on a combined DNA matrix of rbcL, matK and ITS2 genomic regions, retrieved the latter genus as a monophyletic group (Cruz-López et al., 2019) sister to the rest of Cremnophila, Echeveria, Graptopetalum, and some Sedum and Thompsonella species. Another significant result of this work indicated that Pachyphytum cuicatecanum (J.Reyes, Joel Pérez & Brachet) Kimnach is closely related to several Echeveria species (E. penduliflora and others) and clearly do not belong to Pachyphytum, despite sharing many morphological character states. Nevertheless, low resolution problems do not allow the identification of groups (sections) inside Pachyphytum. Previous studies on the “Echeveria Group” (Carrillo-Reyes et al., 2009; Vázquez-Cotero et al., 2017) also recovered Pachyphytum as a monophyletic group, resting on partial species sampling. Historically, the placement of taxa in infrageneric categories (sections) has been complicated, because there is often little congruence between morphological, biogeographic and DNA datasets; this discrepancy between different sources of evidence may have contrasting explanations (e.g. recent diversification vs. decoupling between molecular and morphological evolution).

Table 1.

GenBank accession numbers for the taxa sampled.

The most outstanding character states of Pachyphytum are: presence of membranous scale-like structures known as appendages on the inside of every petal, one on each side of the epipetalous filaments although, some Echeveria species also possess appendages (E. heterosepala, E. longissima var. longissima, E. dactylifera, E. purhepecha, E. rulfiana, among many others); pronounced succulence of the leaves; and pendant scorpioid cymose inflorescences (Reyes et al., 2007). At present, the genus consists of 24 described species — based on morphological, phenological, ecological and geographical distribution aspects — all endemic to Mexico and distributed mainly in central and western regions of this country.

In 2003, a collection of living plants of the family Crassulaceae (Saxifragales: Rosidae) was started at the Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México. In 2005, the Mexican Association of Botanical Gardens (Asociación Mexicana de Jardines Botánicos) declared this collection as “Colección Nacional” (National Collection), representing 70% of the Crassulaceae species known in Mexico. For the establishment of this national collection program, botanical explorations were necessary, as well as the cultivation and maintenance of species of different genera with a georeferenced database. During fieldwork, the genus Pachyphytum received primary attention. The work with the collection has received a great impulse from Christian Brachet, member of the Mexican Cactus Society (Sociedad Mexicana de Cactología, A.C.), who has provided significant help with the logistics of the explorations and with finding information about the type localities of the known species.

The present paper derives from the “Crassulaceae of Mexico” (“Crasuláceas de México”) long-term research project, based on the National Crassulaceae Collection and hosted by JB-IBUNAM. A primary aim of this project has been the gradual construction of a DNA barcoding reference library for Mexican Crassulaceae, to be linked with other datasets in an integrative taxonomy (DeSalle et al., 2005; Goldstein and DeSalle, 2011) framework. Characterbased methods, such as CAOS (Sarkar et al., 2008), have been given preference to establish DNA barcodes, as they allow the definition of discrete DNA diagnostics compatible with apomorphic, morphological character states.

The species described here was first located near La Presita at the base of the Sierra de Santa Bárbara, Guanajuato, in May of 2004 by the team of Christian Brachet, Joel Pérez Crisanto, Roxana Mondragón, and Jerónimo Reyes Santiago. The discovery was made during a trip to locate populations of Echeveria calderoniae PerezCalix growing in the same mountains. At the time of collection, the plants were flowering. The exact locality where the specimens were found is a ravine with high amounts of humidity during the rainy season. Recently, Julia Etter and Martin Kristen have sent specimens of Pachyphytum sp. with identical morphological characteristics from the states of Aguascalientes, Jalisco and Zacatecas to the JB-IBUNAM, and have also shared excellent photos of the new species in habitat.

Table 2.

Primer sequences and PCR thermocycler programs used to amplify DNA fragments.

Table 3.

Molecular diagnostic sites summary for Pachyphytum viscidum and the sampled species of Pachyphytum.

MATERIALS AND METHODS

Sampled taxa: 19 known species of Pachyphytum were sampled, including the new species (Table 1). Five species were not sampled: P. coeruleum J. Meyrán, P. confusum Pérez-Calix, Guadián-Marín & I. García, P. cuicatecanum (J. Reyes, Joel Pérez & Brachet) Kimnach, P. neglectum and P. rogeliocardenasii Pérez-Calix & R. Torres. Regarding P. cuicatecanum, an independent phylogenetic analysis indicates that it does not belong to Pachyphytum (Cruz-López et al., 2019). Due to their recent description, materials were not available for P. coeruleum, P. confusum, P. neglectum, and P. rogeliocardenasii. At least one specimen of the sampled Pachyphytum species was used in molecular analysis.

DNA extraction, amplification and sequencing: Total DNA was extracted from silica-gel dried tissue using the 2X CTAB protocol [standard method at the Canadian Centre for DNA Barcoding (CCDB, University of Guelph, Ontario, Canada)]. DNA fragment amplifications were carried out using Platinum^® Taq DNA Polymerase (Ivanova et al., 2006). Pairs of primers for the three regions selected for amplification and the PCR thermocycler programs used are summarized in Table 2, according to CCDB standards. For rbcL and ITS2 sequences, amplification was performed in 12.5 µL final volumes with 6.25 µL of 10% trehalose. For matK, 1.875 µL of 20% trehalose was used. PCR products were visualized in pre-cast agarose E-gel^® 96 units from Invitrogen. DNA fragments were sequenced at the CCDB facilities. Contig assembly and sequence edition was carried out in Geneious v. R9 ( http://www.geneious.com; Kearse et al., 2012) and submitted to BOLD Systems (Barcode Of Life Database,  http://www.boldsystems.org/). Accession numbers and BOLD Systems IDs for all sequences are provided in Table 1.

Molecular diagnostics analysis: Aligned DNA sequences for the selected markers were downloaded from BOLD Systems. In the alignments, each sequence per species was inspected with PhyDE v0.9971 ( http://www.phyde.de/) and/or analyzed with CAOS (Sarkar et al., 2008), in order to identify DNA diagnostic character states in homologous nucleotide sites per species. These molecular diagnostics were selected and summarized (in order) in Table 3.

RESULTS

DISCUSSION

Although phylogenetic relationships in Pachyphytum are not clearly established, P. viscidum is placed in the sect. Diotostemon (Salm-Dyck) E. Walther based on morphological affinities. In terms of overall appearance, the collected plants are similar to P. hookeri, a species abundant in the area. Furthermore, they are also related to P. brevifolium which also occurs in the state of Guanajuato. However, on closer inspection P. viscidum is distinguished from the two latter ones because its leaves and stems are glutinous, and by the absence of wax (farina) on the leaves. Furthermore, the plants are dark green colored, and the inflorescence born very close to the apical meristem giving the impression of a terminal inflorescence. The new species shows similarities to P. hookeri (Salm-Dyck) A. Berger (Moran, 1990) regarding the shape and coloration of sepals and petals (Fig. 3), but differs by its sticky stems and leaves, dark green thin leaves without wax (Table 4). P. viscidum can also be related to P. brevifolium Rose (Moran, 1963; Pérez-Calix & Glass, 1999) because of its oblanceolate leaves, it is however differentiated from the latter in having longer flowers with narrowed sepals as well as pale pinkish petals and green styles and stigmas (Fig. 3). In short, twentyeight apomorphies were found among the taxa analyzed (Table 4).

Pachyphytum viscidum is one of the species in the genus Pachyphytum that shows molecular diagnostic characters in accordance with character-based DNA barcoding. Referencing the alignment generated in the Barcode of Life Database (BOLD systems), diagnostic characters for P. viscidum correspond to positions 754 and 820 of matK with a G in contrast to the A of P. brevifolium and P. hookeri and the rest of the Pachyphytum species; in turn for region two of the intergenic spacer (ITS2), P. viscidum can also be differentiated from all Pachyphytum species sampled by two DNA diagnostics in positions 298 and 439, both with the T nucleotide, in contrast to the presence of C in the rest of the sequences of all other Pachyphytum species (Table 3).

The description of Pachyphytum viscidum is further supported under integrative taxonomy (sensu DeSalle et al., 2005) criteria: there are both morphological and molecular diagnostic character states associated with the specimens analyzed. Regarding the two genomic regions where DNA diagnostics were found for the new Pachyphytum species, they are currently included in the group of revised core and supplementary loci to perform DNA barcoding in plants (CBOL Plant Working Group, 2009; Hollingsworth, 2011). The use of these two sources of characters as a whole for the description of P. viscidum is enabled by the operational diagram of integrative taxonomy, called “taxonomic circle” by DeSalle et al. (2005; see also Goldstein & DeSalle, 2011). According to the taxonomic circle's different options proposed by DeSalle et al. (2005), the case of P. viscidum corresponds with “sympatric species” where the combination of morphological and molecular evidence supports a new species status for the collected specimens, although they cooccur geographically with other species.”

Figure 1.

Pachyphytum viscidum sp. nov. A. Habit (Photo: J. Etter & M. Kristen). B. P. viscidum in habitat (Photo: J. Etter & M. Kristen). C. Apical rosette detail. D. Detail of stem. E. Detail of leaves. F. Bracteoles in the inflorescence. Photos were taken from Santa Bárbara collection (Guanajuato).

Figure 2.

Pachyphytum viscidum sp. nov. A. Scorpioid cyme. B. Lateral view of the flower. C. Front view of the flower. D. Dissected flower to show stamens and carpels. E. Inner petal appendages. F. Fruit. Photos were taken from Santa Bárbara collection (Guanajuato).

Additional specimens examined: P. brachetii. Mexico. Hidalgo: Mpio. Actopan, 5 km al sur de Actopan, J. Reyes 4350 (MEXU). P. bracteosum. Mexico. Hidalgo: Laguna de Meztitlán, J. Reyes 6275 (MEXU). P. brevifolium. Mexico. Guanajuato: Los Picones, J. Reyes 5048 (MEXU). P. caesium. Mexico. Aguascalientes: Barranca Montoro, J. Reyes 5185 (MEXU). P. compactum. Mexico. Querétaro: La Cañada, J. Reyes 5273 (MEXU). P. contrerasii. Mexico. Jalisco: Mirador Dr. Atl, J. Reyes 5627 (MEXU). P. fittkaui. Mexico. Guanajuato: Los Jofres, J. Reyes 5237 (MEXU). P. garciae. Mexico. Querétaro: El Zapote, J. Reyes 5885 (MEXU). P. glutinicaule. Mexico. Hidalgo: Mpio. Tasquillo, Puerto Juárez, J. Reyes 5653 (MEXU). P. hookeri. Mexico. Guanajuato: Cerro Gordo, J. Reyes 5036 (MEXU). P. kimnachii. Mexico. San Luis Potosí: Cerro Agujón, J. Reyes 5305 (MEXU). P. longifolium. Mexico. Hidalgo: entre Gilo y Almolon, J. Reyes 6276 (MEXU). P. machucae. Mexico. Michoacán: Cañada de Agua, J. Reyes 5071 (MEXU). P. oviferum. Mexico. San Luis Potosí: El Cajón, J. Reyes 6038 (MEXU). P. rzedowskii. Mexico. Michoacán: 1.5 km al noroeste de Puente Grande, J. Reyes 5562 (MEXU). P. saltense. Mexico. Zacatecas: El Salto, J. Reyes 3644 (MEXU). P. viscidum. (paratypes). Mexico. Aguascalientes: Mpio. Tepezala, in cliffs above RMO Tepezala, 2421 m, June 16, 2012, Julia Etter & Martin Kristen 3619 (MEXU). Jalisco: Mpio. Ojuelos de Jalisco, 3 km NW of Matanzas, between Lagos de Moreno and Ojuelos along highway 80, in cliffs behind the cemetery, 2320 m, March 27, 2012 (flowering), Julia Etter & Martin Kristen 3494 (MEXU). Zacatecas: Mpio. Villa García, 4.5 km from Villa García along dirt road to Loreto, 2150 m, May 15, 2012, Julia Etter & Martin Kristen 3531 (MEXU). P. viride. Mexico. Guanajuato: entrada al Pueblo de Coconoxtle, J. Reyes 5873 (MEXU). P. werdermannii. Mexico. Tamaulipas: Microondas Las Mulas, Julia Etter & Martin Kristen 2000.

Figure 3.

Morphological comparisons between Pachyphytum viscidum sp. nov. and its closely related species. A. P. viscidum (from Santa Bárbara, Guanajuato), B. P. brevifolium (from Los Picones, Guanajuato). C. P. hookeri (from Cerro Gordo, Guanajuato).

Table 3.

Comparison of morphological character states between Pachyphytum brevifolium, P. hookeri and P. viscidum sp. nov.