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Unravelling the Mexican Magnolia dealbata (Magnoliaceae) species complex

February 2025
Phytotaxa 684(1):1-32

DOI:10.11646/phytotaxa.684.1.1

Authors:

Fabián Augusto Aldaba Núñez

Institute of Ecology INECOL

Salvador Guzmán-Díaz

Institute of Ecology INECOL

Show all 6 authorsHide

In the last two decades, approximately 80 new Magnolia species have been described from the Neotropics; thus this region now hosts almost half of the world’s known Magnolia diversity. Many of these likely are not segregate taxa but rather separate populations or groups of populations of the previously broadly circumscribed, widespread species. Such is possibly the case of the Magnolia dealbata species complex (belonging to Magnolia sect. Macrophylla), distributed throughout the Sierra Madre Oriental mountain range in Eastern Mexico. This species complex has been divided into six morphospecies based on morphological criteria only. However, recent microsatellite markers have suggested that these may be a single entity. Considering geographical data and the isolation of populations, we hypothesised that the different morphospecies could form two entities, corresponding to the north and centre of the Sierra Madre Oriental. This hypothesis was tested by morphological observations, chloroplast comparisons and phylogenetic analyses of plastomes, angiosperm DNA plastid barcodes and Magnolia-specific plastid DNA barcodes from hypervariable regions. Phylogenetic results from plastomes and angiosperm DNA plastid barcodes refute the multispecies hypothesis and show that the six morphospecies of this complex inhabiting the Sierra Madre Oriental form a single entity. Evidence is also provided that the morphological characters used to delimit the morphospecies of the complex, mainly numbers of carpels and the absence-presence and colour of a spot in the petals, are, in fact, phenotypic variation and have no taxonomic significance. Therefore, the taxa M. alejandrae, M. nuevoleonensis, M. rzedowskiana, M. vovidesii and M. zotictla are synonymised here under M. dealbata. However, the possibility remains of including the latter as a variety of M. macrophylla, based on the results of the Magnolia-specific plastid DNA barcodes. Furthermore, this study proposes an updated conservation status for M. dealbata, highlighting the urgent need for effective conservation measures. The taxonomic clarification presented here is essential to properly target such efforts, especially in the face of threats such as indiscriminate collection and vulnerability to environmental disturbance.

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Phytotaxa 684 (1): 001–032

https://www.mapress.com/pt/

ISSN 1179-3155 (print edition)

ISSN 1179-3163 (online edition)

Accepted by Eduardo Cires Rodríguez: 3 Jan. 2025; published: 5 Feb. 2025

https://doi.org/10.11646/phytotaxa.684.1.1

Licensed under Creative Commons Attribution-N.C. 4.0 International https://creativecommons.org/licenses/by-nc/4.0/

Unravelling the Mexican Magnolia dealbata (Magnoliaceae) species complex

FABIÁN AUGUSTO ALDABA NÚÑEZ1,5,*, SALVADOR GUZMÁN-DÍAZ1,6, SUHYEON PARK2,7, SANGTAE

KIM2,8, ESTEBAN MANUEL MARTÍNEZ SALAS3,9 & MARIE-STÉPHANIE SAMAIN1,4,10

1Red de Diversidad Biológica del Occidente Mexicano, Instituto de Ecología, A.C., Avenida Lázaro Cárdenas 253, 61600, Pátzcuaro,

Michoacán, Mexico

2Department of Biology, Sungshin Women’s University, 76 Ga-gil 55, Gangbuk-gu, Seoul, 01133, Republic of Korea

3Herbario Nacional de México, Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Circuito

Zona Deportiva, 4510, Mexico City, Mexico

4Botanical Garden, Ghent University, K.L. Ledeganckstraat 35, 9000, Gent, Belgium

�

fabian.aldaba@outlook.com; https://orcid.org/0000-0002-0271-4467

�

guzmandsalvador@gmail.com; https://orcid.org/0000-0002-5123-1197

�

shpark817@naver.com; https://orcid.org/0000-0002-3321-1950

�

amborella@sungshin.ac.kr; https://orcid.org/0000-0002-1821-4707

�

ems@ib.unam.mx; https://orcid.org/0000-0003-3882-899X

�

mariestephanie.samain@gmail.com; https://orcid.org/0000-0002-7530-9024

*Corresponding author:

�

fabian.aldaba@outlook.com

Abstract

In the last two decades, approximately 80 new Magnolia species have been described from the Neotropics; thus this region

now hosts almost half of the world’s known Magnolia diversity. Many of these likely are not segregate taxa but rather

separate populations or groups of populations of the previously broadly circumscribed, widespread species. Such is possibly

the case of the Magnolia dealbata species complex (belonging to Magnolia sect. Macrophylla), distributed throughout the

Sierra Madre Oriental mountain range in Eastern Mexico. This species complex has been divided into six morphospecies

based on morphological criteria only. However, recent microsatellite markers have suggested that these may be a single entity.

Considering geographical data and the isolation of populations, we hypothesised that the different morphospecies could form

two entities, corresponding to the north and centre of the Sierra Madre Oriental. This hypothesis was tested by morphological

observations, chloroplast comparisons and phylogenetic analyses of plastomes, angiosperm DNA plastid barcodes and

Magnolia-specific plastid DNA barcodes from hypervariable regions. Phylogenetic results from plastomes and angiosperm

DNA plastid barcodes refute the multispecies hypothesis and show that the six morphospecies of this complex inhabiting

the Sierra Madre Oriental form a single entity. Evidence is also provided that the morphological characters used to delimit

the morphospecies of the complex, mainly numbers of carpels and the absence-presence and colour of a spot in the petals,

are, in fact, phenotypic variation and have no taxonomic significance. Therefore, the taxa M. alejandrae, M. nuevoleonensis,

M. rzedowskiana, M. vovidesii and M. zotictla are synonymised here under M. dealbata. However, the possibility remains

of including the latter as a variety of M. macrophylla, based on the results of the Magnolia-specific plastid DNA barcodes.

Furthermore, this study proposes an updated conservation status for M. dealbata, highlighting the urgent need for effective

conservation measures. The taxonomic clarification presented here is essential to properly target such efforts, especially in

the face of threats such as indiscriminate collection and vulnerability to environmental disturbance.

Key words: Barcodes, Conservation status, Macrophylla, Plastome, Sierra Madre Oriental

Introduction

The infrageneric classification of Magnolia Linnaeus (1753: 535) subdivides the genus into 15 sections (Wang et

al. 2020; Aldaba et al., in prep.) comprising a total of ca. 400 species distributed mainly in temperate and tropical

mountainous areas of Asia and the Americas (Meyer 1993; POWO 2023; TROPICOS 2023; Xia et al. 2008). One of

these sections is the North American endemic clade Magnolia sect. Macrophylla Figlar & Nooteboom (2004: 92), the

species of which are morphologically distinguished by being deciduous trees, the presence of false leaf whorls on new

growth branches, generally large (>20 cm) and auriculiform leaves with auriculate or cordate base, pulverulent abaxial

ALDABA ET AL.

surface, stipules semi-attached to the petiole and leaving a scar on it, and rather large flowers (>25 cm), usually with

a purple or orange spot at the base of the adaxial side of the petals. The morphologically most similar clade is the

Magnolia sect. Tuliparia Spach (1839: 477; Magnolia sect. Auriculata Figlar & Nooteboom (2004: 92) sensu Figlar &

Nooteboom 2004), with only two known varieties of a single species: M. fraseri Walter (1788: 159) var. fraseri and M.

fraseri var. pyramidata (W.Bartram) Pampanini (1915: 230)., inhabiting the Appalachian region (Meyer 1993; Wang

et al. 2020).

The main distinction between the two sections is the pubescence on the abaxial leaf surface of the taxa in Magnolia

sect. Macrophylla, which is absent in Magnolia sect. Tuliparia (Meyer 1993). Phenologically, differences in floral

odours and anthesis have also been observed, with the Magnolia sect. Tuliparia flowers closing completely at night

(Figlar 2019; Thien 1974). However, molecular evidence provides varying insights into the relationships of Magnolia

sect. Macrophylla: Plastid data confirm Magnolia sect. Tuliparia as its sister group, while nuclear data suggest that

its closest relative is Magnolia sect. Magnolia; the latter group also inhabits the SMOr and other mountain ranges in

Mexico (Aldaba Núñez et al. 2024).

Magnolia sect. Macrophylla includes nine currently accepted species. Of these, seven are endemic to Mexico

(García-Morales et al. 2017; Sánchez-González et al. 2021; Vázquez-García et al. 2013, 2015, 2016, 2021; Zuccarini

1837). Six species are distributed along the Sierra Madre Oriental (SMOr): M. alejandrae García-Morales & Iamonico

(2017: 239) occurs in the northern SMOr (central-western Tamaulipas state); M. dealbata Zuccarini (1837: 373) in the

southern SMOr (central Oaxaca state); M. nuevoleonensis A.Vázquez & Domínguez-Yescas (2016: 49) in the extreme

north of the SMOr (central Nuevo León state); M. rzedowskiana (A.Vázquez, Domínguez-Yescas & R.Pedraza 2015:

23) in the north-central SMOr (Hidalgo, Querétaro, and San Luis Potosí states); M. vovidesii (A.Vázquez, Domínguez-

Yescas & L.Carvajal 2013: 478) in the south-central SMOr (Veracruz state); and M. zotictla A.Sánchez-González,

Gutiérrez-Lozano & A.Vázquez (2021: 272) in the central SMOr (Hidalgo and Puebla states). One species, M. mixteca

A. Vázquez& Domínguez-Yescas (2021: 202), is found in the Sierra Madre del Sur (SMS). The remaining two species

are found in the Appalachians, in the southeastern United States of America (Meyer 1993): M. ashei Weatherby (1926:

35) and M. macrophylla Michaux (1803: 327).

The taxa from Magnolia sect. Macrophylla are known for their diverse insecticidal and medicinal applications,

such as an analgesic, antidiarrheal, gastrointestinal aid, respiratory aid, and toothache remedy, some of which have

been documented since pre-Hispanic times (Alonso-Castro et al. 2011, 2014; Chen et al. 2021; Domínguez et al. 2009,

2010, 2016; Domínguez-Yescas & Tapia 2013; Flores-Estévez et al. 2013; González-Trujano et al. 2015; Guzmán

Gutiérrez et al. 2014; Guzmán-Trampe et al. 2015; Jacobo-Salcedo et al. 2011; Martínez et al. 2006; Moerman 1991;

Ramírez-Reyes et al. 2015; Rauf et al. 2021; Rodríguez-Ramírez et al. 2021).

Among the Mexican species of Magnolia sect. Macrophylla, only M. dealbata was originally known to be

distributed throughout the SMOr, especially montane cloud forests, one of the most threatened ecosystems in Mexico

(Gual-Díaz & Rendón-Correa 2017; Miranda & Hernández-X. 2014; Rzedowski 2006; Williams-Linera et al. 2016b).

This species was long believed to be extinct (Gutiérrez Carvajal 1993; Vovides 1981); however, since 2013, different

populations (or localities disjointed by at least 70 km) throughout the central part of the SMOr have been segregated

as new species: M. rzedowskiana, M. vovidesii, and M. zotictla (García-Morales et al. 2017; Sánchez-González et al.

2021; Vázquez-García et al. 2013, 2015, 2016, 2021).

Those new species have been described based on a few specimens (1 to 13 vouchers), and the morphological

characters used to define them mostly considered colours (fruits, flowers), sizes (stamens, gynoecium, leaves, petals,

size) and numbers of floral structures (carpels, stamens), which are highly variable traits in angiosperms (Basnett

et al. 2024; Dai et al. 2016; Delgado et al. 2021; Zu et al. 2020) and in Magnolia (Aldaba Núñez 2020). Moreover,

their delimitation does not integrate data on genetics, geography, biogeography, or geology, only a few differences

in vegetation. Hence, the delimitation of these new species needs further investigation, especially in light of the

development of conservation actions, but also because of their enormous potential in medicinal applications.

The existence of a species complex comprising these three taxa from the central SMOr segregated from M. dealbata

and two others from the northern SMOr (M. alejandrae and M. nuevoleonensis) has been suggested based on genetic

data of a population genetics study for the conservation of these species. In this work, Chávez-Cortázar et al. (2021)

showed, using microsatellite markers, that all the species of the Magnolia dealbata complex were not reflected in the

analyses of genetic structure, while the greatest genetic variability was found between groups of populations instead

of groups of species. It was therefore concluded that gene pools may instead represent well-differentiated populations

of a single widely distributed species: M. dealbata. This statement is also supported by preliminary morphological

observations, where the observed differences in colour, size, and number of parts may be intraspecific population

variation along a north-south latitudinal gradient.

In addition to the genetic data, observations of phenotypic plasticity, both inter- and intraspecific, such as those

realized by Gutiérrez-Lozano et al. (2020) and Rodríguez-Ramírez et al. (2021) confirm the existence of this species

complex. The first study focused on the morphology (using 26 traits) of four populations of M. rzedowskiana and

found a significant variation in leaves and flowers. The second study analysed the leaf vein morphological variation

in four species (M. alejandrae, M. nuevoleonensis, M. vovidesii, and M. rzedowskiana), using four traits, and found

no significant differences between them. Subsequently, (Aldaba Núñez et al. 2024) conducted a study of Neotropical

magnolias using morphological, nuclear genomic and plastome data. Following these analyses, the taxa from Magnolia

sect. Macrophylla did not exhibit a consistent pattern in the phylogenetic trees; although they conform a monophyletic

group, the samples appeared scattered across various branches of the trees, indicating a lack of clear lineage grouping.

In addition, several polytomies were observed, complicating the understanding of the phylogenetic relationships

among the taxa. Moreover, many of the clades recovered had low support values (< 0.5), showing the absence of

strong evidence for these groupings.

A similar pattern has previously been found in Magnolia sect. Talauma (Juss.) Baillon (1868: 142), where the

microsatellite markers together with morphological observations of M. lopezobradorii A.Vázquez (2012: 110) and

M. zoquepopolucae A.Vázquez (2012: 52 ; two species with doubtful taxonomic delimitation segregated from M.

mexicana Candolle (1818: 451) showed that both conform to a single taxon (Aldaba Núñez et al. 2021). Similarly, in

Magnolia sect. Magnolia, chloroplast and nuclear DNA sequences, as well as microsatellite markers, did not support

the genetic differentiation of M. pedrazae A.Vázquez (2013: 475)which had been segregated from M. schiedeana

Schlechtendal (1864: 144; Rico & Becerril 2019).

Accurate species delimitation within the Magnolia dealbata complex is crucial for conservation efforts. Although

there are already several conservation studies on some of the taxa, if their taxonomic delimitation is not clear or without

a clear understanding of the species diversity within the complex, the conservation strategies may be ineffective and

would hinder their management (Corral-Aguirre & Sánchez-Velásquez 2006; Galván-Hernández et al. 2020; García-

Hernández & Toledo-Aceves 2020; Gutiérrez Carvajal 1993; Gutierrez & Vovides 1997; Martínez et al. 2006; Ramírez-

Bamonde et al. 2005; Sánchez-Velásquez et al. 2016; Sánchez‐Velásquez & Pineda‐López 2010; Sánchez-Velásquez

& Pineda-López 2006; Smit 2013; Velazco-Macías et al. 2008; Vovides & Iglesias 1996). Furthermore, the taxa within

the complex inhabit highly threatened cloud forests (Domínguez-Yescas et al. 2020).

Therefore, the work aimed to resolve the species complex present in the Mexican species of the Magnolia sect.

Macrophylla by morphological observations and molecular analyses. The main questions addressed in this study were:

1) How many species of Magnolia sect. Macrophylla are found in the Sierra Madre Oriental in Mexico?, 2) What

are the species’ taxonomic limits based on morphological and molecular data?, 3) To what extent are the characters

(reproductive and vegetative) used for species delimitation within the Magnolia dealbata complex taxonomically

reliable? and 4) How do they relate to each other phylogenetically? Our hypothesis, based on preliminary taxonomic

analyses as well as previous molecular and morphological work, is that the six-species complex of M. dealbata from

the SMOr constitute at least two entities: 1) The northern SMOr species: M. alejandrae and M. nuevoleonensis and 2)

The central-south SMOr species: M. dealbata, M. rzedowskiana, M. vovidesii, and M. zotictla.

Materials and methods

Field sampling

Field sampling was conducted in the SMOr, focusing on regions known to harbour taxa from the M. dealbata complex.

The sampling process took place over two years, from 2014 to 2015. During this period, approximately 2.5 cm portions

of young leaves were collected and preserved in silica gel for subsequent laboratory analyses. They were labelled with

field information such as the identity, date of collection, geographical location, and observed phenotypic characteristics

(flowering or fruiting).

Furthermore, four molecular samples of the study taxa, collected and provided by Sangtae Kim (BioProject ID:

PRJNA994423), as part of a project to study evolutionary relationships in the Magnoliaceae Jussieu (1789), were used

to reinforce the sampling. We included at least one sample per taxon, comprising in total 14 molecular samples from

nine taxa (all six morphospecies in the Magnolia dealbata complex, the two US taxa from Magnolia sect. Macrophylla

and M. fraseri from Magnolia sect. Tuliparia), with each sample representing one different population covering the

full distribution of the species complex. Detailed sampling information can be found in Table 1.

ALDABA ET AL.

TABLE 1. Sampling list of the 14 North American Magnolia taxa studied. Note: *: Transplanted or seedlings provided by Wisley

Garden.

Taxa Lab ID Section Field origin Voucher (herbarium) GenBank accession

number

M. alejandrae García-Mor. &

Iamonico MA1160A Macrophylla Mexico, Tamaulipas Mata 1160 (XAL) OR730681

M. alejandrae MA1188B Macrophylla Mexico, Tamaulipas Mata 1188 (XAL) OM455404

M. ashei Weath. MA0016 Macrophylla *South Korea, Chungcheongnam-do

(Chollipo arboretum) Kim 1016 (NPRI) PQ842611

M. cf. dealbata MA0008 Macrophylla *South Korea, Chungcheongnam-do

(Chollipo arboretum) Kim 1008 (NPRI) OR730743

M. dealbata Zucc. MA3190 Macrophylla Mexico, Oaxaca Mata 807 (XAL) OR730679

M. fraseri Walter MA0291 Tuliparia *South Korea, Chungcheongnam-do

(Chollipo arboretum) Kim 1111 (NPRI) OR730746

M. macrophylla Michx. MA0015 Macrophylla *South Korea, Chungcheongnam-do

(Chollipo arboretum) Kim 1015 (NPRI) PQ842612

M. nuevoleonensis A.Vázquez &

Domínguez-Yescas MA1138A Macrophylla Mexico, Nuevo León Mata 1138 (XAL) OR730704

M. rzedowskiana A.Vázquez,

Domínguez-Yescas & R.Pedraza MA1004A Macrophylla Mexico, Querétaro Mata 1004 (XAL) OR730716

M. rzedowskiana MA3188B Macrophylla Mexico, San Luis Potosí Mata 1118 (XAL) OR730717

M. vovidesii A.Vázquez,

Domínguez-Yescas & L.Carvajal MA0092B Macrophylla Mexico, Veracruz

Chávez-Cortázar &

Hernández-Sánchez 92

(XAL)

OR730729

M. vovidesii MA0624B Macrophylla Mexico, Veracruz

Chávez-Cortázar &

Hernández-Sánchez

624 (XAL)

OR730730

M. vovidesii MA0877A Macrophylla Mexico, Veracruz Mata 877 (XAL) OR730680

M. zotictla A.Sánchez-Gonz.,

Gut.-Lozano & A.Vázquez MA0866A Macrophylla Mexico, Puebla Mata 866 (XAL) OM455410

Morphological observations

Morphological analyses were conducted at population and morphospecies level to gain insights into the variations

within the M. dealbata complex. A total of 168 vouchers (including the types) were consulted from 26 herbaria,

both physically and through their digital collections: BR, BRIT, F, HUAP, IBUG, IEB, ITCV, K, LL, LSU, M, MA,

MEXU, MO, NCU, NCSC, NY, P, PH, TEX, US, USCH, URV, VT, WAG, XAL (Herbarium names according to

Thiers continuously updated). Field pictures from iNaturalist.org (whose taxonomic identity was verified) were also

examined. The protologues of each taxon were also considered and revised (García-Morales et al. 2017; Sánchez-

González et al. 2021; Vázquez-García et al. 2013, 2015, 2016, 2021; Zuccarini 1837).

All specimens and pictures underwent detailed morphological observations. From the visual evaluation of the

phenotypic characteristics of the specimens, a list of 13 characters was compiled (table 2). The botanical terms of the

character states were standardized following the proposal of Moreno (1984).

Preliminary conservation status assessment

We conducted a preliminary assessment of the conservation status of the taxa based on the morphological observations

and the molecular and phylogenetic results. Our methodology involved field surveys to collect data on species

distribution, population size, and habitat conditions. We also reviewed historical records and consulted with local

experts to gather additional insights. The criteria set forth by the International Union for Conservation of Nature

(IUCN) Red List (IUCN 2013, 2021; IUCN Standards and Petitions Committee 2024) were applied to evaluate the risk

of extinction faced. This included analysing factors such as geographic range, population trends, and threats to habitat

integrity. The online tool GeoCAT (Bachman et al. 2011) was used to calculate the Extent of Occurrence (EOO) and

the Area of Occupancy (AOO), using the data from vouchers and carefully curated GBIF samples.

TABLE 2. Traits for the Magnolia sect. Macrophylla taxa occurring in the Sierra Madre Oriental in Mexico.

Taxa Indumentum of

young branches

Indumentum of

young petioles

Indumentum

of stipule Leaf shape Leaf base shape Leaf apex

shape

Under

surface veins

indumentum

Peduncle

indumentum

Bract

indumentum

Indumentum

of gynoecium

Fruit

shape

Fruit

indumentum

Beaked

mature

follicles

Magnolia alejandrae García-Mor.

& Iamonico Pulverulent Glabrous Pubescent Auriculiform Auriculate, Cordate Acute Pulverulent Pulverulent Pulverulent Glabrous Ovoid Glabrous Absent

M. dealbata Zucc. Pulverulent Pulverulent Pubescent Auriculiform Auriculate, Cordate Acute Pulverulent Pulverulent Pulverulent Glabrous Ovoid Glabrous Absent

M. nuevoleonensis A.Vázquez &

Domínguez-Yescas Pulverulent Glabrous Glabrous Auriculiform Auriculate, Cordate Acute Pulverulent Pulverulent Pulverulent Tomentose Ovoid Glabrous Absent

M. rzedowskiana A.Vázquez,

Domínguez-Yescas & R.Pedraza Sericeous Pulverulent Pubescent Auriculiform Auriculate, Cordate Acute Pulverulent Pulverulent Pulverulent Tomentose Ovoid Glabrous Absent

M. vovidesii A.Vázquez,

Domínguez-Yescas & L.Carvajal Pulverulent Glabrous Pubescent Auriculiform Auriculate, Cordate Acute Pulverulent,

Sericeous Pulverulent Pulverulent Glabrous Ovoid Glabrous Absent

M. zotictla A.Sánchez-Gonz., Gut.-

Lozano & A.Vázquez Sericeous Pulverulent,

Sericeous Pubescent Auriculiform Auriculate, Cordate Acute Pulverulent Glabrous,

Pulverulent Pulverulent Glabrous Ovoid Glabrous,

Sericeous

Absent,

Present

ALDABA ET AL.

DNA extraction and sequencing

A modified CTAB method (Larridon et al. 2015) was utilized for the DNA extraction process. The quality of the

extracted DNA was then evaluated using a Nanodrop 2000 UV-Vis spectrophotometer. Subsequently, the samples were

dispatched to RAPiD Genomics located in Gainesville, Florida, US, for sequencing. The sequencing was carried out

using the Genome Skimming technique to compile complete chloroplast genomes.

Plastome assembly and annotation

A first quality check of the demultiplexed samples was performed using the software FastQC v. 0.11.7 (Andrews

2019). Trimmomatic v. 0.38 (Bolger et al. 2014) was used to filter low-quality reads and perform the adapter trimming,

applying a sliding window of 5:20 and removing all the reads shorter than 30 bases.

GetOrganelle v. 1.7.0 (Jin et al. 2020) was used to assemble the plastome; this is a complete Python software

that uses Illumina reads to perform de novo plastome assemblies. This software makes use of Bowtie2 (Langmead &

Salzberg 2012), SPAdes (Bankevich et al. 2012), BLAST (Camacho et al. 2009) and Biopython (Cock et al. 2009).

The program “Get_organelle_from_reads.py” was used with the “embplant_pt” option, as well as 15 extension rounds

and kmer values between 21 and 105. The results were visualized with Bandage v. 0.8.1 (Wick et al. 2015) to ensure

that a correct assembly graph was produced.

The plastome annotation was performed using the online software GeSeq v. 1.55 (https://chlorobox.mpimp-golm.

mpg.de/geseq.html) (Tillich et al. 2017). Chloë v. 0.1.0 (https://chloe.plantenergy.edu.au/;unpublished) was used as

a support annotator, and ARAGORN v. 1.2.38 (Laslett & Canback 2004) and tRNAscan-SE v. 2.0.7 (Chan & Lowe

2019) were used as tRNA annotators, keeping the best annotation only. As a reference for the annotation, we utilized

the base MPI-MP reference set, as well as the Magnoliaceae plastomes available at the NCBI RefSeq (O’Leary et al.

2016).

Plastome comparative analysis

All assembled plastomes and their annotations were submitted to mVista (Frazer et al. 2004). We used the Shuffle-

LAGAN mode (Brudno et al. 2003) to align the sequences and perform pairwise comparisons. Next, the plastomes

were aligned using Mauve v. 2.4.0 (Darling et al. 2004), with the progressive alignment option, and the gene orders

were compared.

DNAsp v. 6.12.03 (Librado & Rozas 2009) was used to calculate the nucleotide diversity among the aligned

genomes using a sliding-window approach with a window length of 600 bp and a step size of 200 bp. All the complete

annotated plastomes were submitted to IRscope (Amiryousefi et al. 2018) to analyse the expansion and contraction of

the IR regions.

Phylogenetic analyses

Three different plastid datasets were used independently to elucidate the Magnolia dealbata complex: 1) The complete

plastomes of each sample; 2) The complete sequences of the following genes: matK, rbcL, trnH, psbA and trnL-F,

which have been suggested as plastid barcodes to infer phylogenetic relationships in closely related taxa in angiosperms

(Hu & Wang 2023; Li et al. 2015); and 3) The group-specific DNA barcodes selected from hypervariable regions in

Neotropical Magnolia plastomes, ccsA, ndhD, petL, and rpl32, have been identified in previous studies (Guzmán-Díaz

et al. 2022).

Each dataset was aligned using MAFFT with the default parameters. The MAFFT alignment was used to build

species trees with maximum likelihood (ML) and Bayesian inference (BI). The ML approach was performed in IQ-Tree

v. 2.0.3 (Minh et al. 2020) with an ultrafast bootstrap to estimate the branch support values. The software MrBayes v.

3.2.7 was used for the BI analysis, applying a GTR + I + gamma model with 10 million generations and a burn-in of

25%. Magnolia fraseri from the -2016633688 Magnolia sect. Auriculata was used to root the trees.

Results

Morphological observations

According to the list of 13 characters obtained from the observations of the specimens collected, the 168 herbarium

samples and the photographs taken in the field (Table 2), it has been observed that the taxa of the Magnolia dealbata

complex have a few traits that may allow them to be distinguished. These are mainly the indumentum on the veins of

the abaxial surface of the leaves, as well as differences in the pubescence of the stipules, bracts, and fruits.

However, it is important to emphasize that while certain morphological characteristics were found to be relatively

conserved, they are not sufficient for distinguishing between taxa, as these features varied among individuals from the

same locality (e.g., abaxial leaf surface indumentum in M. vovidesii). Similarly, traits initially identified as unique to

M. zotictla (e.g., sericeous indumentum on young petioles and fruits, glabrous indumentum on peduncles, and beaked

mature follicles) were found to vary among individuals within its populations.

The observed lamina sizes were quite variable and did not show any pattern among the taxa, rather the maximum

leaf lengths overlapped between the taxa. The same was true for the size of the floral parts. However, no length or

width measurements nor counting of parts (number of carpels or stamens) have been carried out as most material was

seen digitally due to the COVID-19 pandemic and because we wanted to test different characters based on shapes

and textures. Nevertheless, we do not consider this to be a problem for our study since we are focusing on variations

in organ shapes, which are proving to be more informative and of greater taxonomic importance, as has already been

confirmed in species from the Magnolia sect. Talauma (Aldaba Núñez 2020).

The most stable or conserved traits among the species complex were those related to leaves shapes (all with acute

apex, auriculate or cordate base and auriculiform lamina), bract indumentum (pulverulent) and fruit shape (ovoid);

while the most variable traits were related to the indumentum of: young branches (from pulverulent to sericeous),

young petioles (from glabrous to sericeous), stipules (glabrous or pubescent), gynoecium (glabrous or tomentose) and

fruits (glabrous or sericeous).

FIGURE 1. Taxa comprising the Magnolia dealbata Zucc. (Magnoliaceae) species complex in the Sierra Madre Oriental in eastern

Mexico.

ALDABA ET AL.

Plastome features and comparative analysis

The Magnolia plastomes ranged in size length from 159 879 bp in M. zotictla (sample MA0866A) to 160 087 bp

in M. macrophylla (sample MA0015) and exhibited a quadripartite structure, including a large single-copy region

(LSC, ranged from 87 967 to 88 175 bp), a small single-copy region (SSC, ranged from 18 735 to 18 787 bp), and

two inverted repeated regions (IRs, ranged from 26 585 to 26 597 bp). The total GC content was 39% for all studied

samples. The Shuffle-LAGAN alignment in mVista resulted in the similarity plot shown in Supplementary file 1;

overall, the Magnolia plastomes presented high similarity values across the whole genome, with small regions of lower

similarity. The Mauve alignment results are presented in Figure 2. One colinear block was identified, while all regions

of the 14 Magnolia plastomes were in the same order and orientation.

FIGURE 2. Mauve progressive alignment, including all 14 North American Magnolia taxa plastomes. Blocks of the same colour linked

by a line represent colinear regions. Blocks below the graphs represent coding regions.

From the sliding window analysis performed in DNAsp, we obtained the nucleotide diversity values (Pi) from all

samples (Fig. 3). These ranged from 0 to 0.0095 and presented a mean of 0.0007. The most diverse sites corresponded

to genes, such as psbJ, petL, rpl22—rps18, ccsA, ndhD, and ycf1; as well as intergenic regions in the LSC and SSC

regions, while the IR regions presented the lowest diversity.

The plastomes of all 14 samples (corresponding to nine taxa) were compared to visualize the overall sequence

divergence (Fig. 4). Comparing the IR/LSC and IR/SSC boundaries in the 14 plastomes uncovered stable IRs with

little expansion or contraction. The LSC-IRb borders were found to be located within the rps19 gene, and the SSC/IRa

was located in the coding region of the ycf1 gene. The IR regions of all taxa were well-conserved, ranging from 25 585

bp in M. macrophylla and M. ashei to 26 597 bp in M. fraseri. The IR regions of almost all Mexican taxa recovered

26 587 bp, except for the samples M. vovidesii MA0877A and M. zotictla, which recovered 26588 bp. The gene

distribution at the four boundaries was the same in all plastomes.

Phylogenetic analyses

Overall, both the Bayesian Inference (BI) and Maximum-likelihood (ML) analyses produced fairly similar topologies

in each of the three molecular datasets analysed (angiosperm DNA barcodes, Magnolia DNA barcodes and Magnolia

plastomes).

In the BI phylogenetic hypothesis based on the complete chloroplasts, the samples of Magnolia sect. Macrophylla

formed two differentiated clades (Fig. 5, Fig. S1). The first consisted of the two US taxa (posterior probability, PP=1),

while the second grouped all Mexican taxa (PP=1). Within the Mexican clade, M. nuevoleonensis was placed as the

sister group to this Mexican clade, which was composed of M. zotictla and all the remaining taxa (PP=0.71), hereafter

referred to as the core group (PP=1). Within this core group, the two samples of M. alejandrae formed a clade separated

from the rest, maintaining high support values between them (PP=1). Within the core group, a three-branch polytomy

was formed consisting of a sample of M. vovidesii (MA0877A), the remaining samples of this taxon together with

those of M. dealbata, and the two samples of M. rzedowskiana, which formed their own clade but with low support

values between them (PP=0.66); while the samples of M. dealbata and M. vovidesii formed a single clade with low

support (PP=0.58) intersecting among them. The same pattern was maintained in the ML-based trees, with the only

difference being that no polytomy was recorded in the core group, but all M. dealbata and M. vovidesii samples were

grouped into a single clade (bootstrap support percentage, BP=47). The BP was also similar to the PP in all clades.

FIGURE 3. Nucleotide diversity values (Pi) resulting from the sliding window analysis of the 14 North America Magnolia plastomes.

The BI phylogenetic hypothesis derived from the DNA barcodes also recovered the two main clades in the tree

based on chloroplast data: the Mexican taxa on the one hand (PP=0.64) and the US taxa on the other (PP=1; Fig. 6, Fig.

S2). However, in this case, there was no resolution, and all Mexican taxa were grouped into a large polytomy. In the

ML tree, a sample of M. vovidesii (MA0877A) behaved as a sister to all other Mexican taxa, including the remaining

samples of M. vovidesii (BP=63). Furthermore, the relationships between the M. alejandrae and M. rzedowskiana

samples were no longer recovered, but the taxa appeared within a large polytomy.

Finally, the topologies obtained from the hypervariable regions were completely different from the two previously

described (Fig. 7, Fig. S3). In the BI tree, two clades were also recovered: on the one hand, taxa from the US (PP=1)

and the northern SMOr (M. alejandrae and M. nuevoleonensis, the latter as sister to the others) and, on the other hand,

the remaining taxa from the central and southern SMOr (PP=0.98), both clades forming polytomies. In the ML tree,

this north-south pattern was maintained, the only difference being that in the northern clade, M. nuevoleonensis was

the sister of all other taxa, while in the southern clade, the sister group was M. zotictla (BP=63).

Taxonomic treatment

Magnolia dealbata Zucc. Abh. Math.-Phys. Cl. Königl. Bayer. Akad. Wiss. 2: 373. 1837. ≡ Magnolia macrophylla

var. dealbata (Zucc.) D.L.Johnson, Baileya 23: 56. 1989. Type:—MEXICO. Oaxaca, prope Rincou [Rincon, Villa

Alta], 600–900 m, Karwinski s.n. [epitype, designated by García-Morales et al. (2017: 242): M239898!; lectotype,

designated by García-Morales et al. (2017: 242): [icon]: “Magnolia dealbata” in Zuccarini, Abh. Math.-Phys. Cl.

Königl. Bayer. Akad. Wiss. 2. Icon. 373: t. 3. 1836 ].

ALDABA ET AL.

= Magnolia alejandrae García-Mor. & Iamonico, Phytotaxa 309(3): 239. 2017. Type:—MEXICO. Tamaulipas: Municipio de Victoria,

Rancho El Molino, bosque mesófilo de montaña, 23°45’56.02’’N 99°19’31.27’’W, 1500 m, 1 May 2016, García-Morales 5435

(holotype: ITCV!; isotypes: GBH, HFLA, HUAP!, ITCV!, MEXU!, SLPM, UAT).

= Magnolia nuevoleonensis A.Vázquez & Domínguez-Yescas, Nordic J. Bot. 34: 49. 2015. Type:—MEXICO. Nuevo León: Municipio de

Montemorelos, Ejido La Trinidad, 25°11’45’’N 100°06’59’’W, 1600 m, 21 Mar 2006, C. G. Velazco-Macías s. n. (holotype: UNL;

isotype: IBUG).

= Magnolia rzedowskiana A.Vázquez, Domínguez-Yescas & R.Pedraza, Acta Bot. Mex. 112: 23. 2015. Type:—MEXICO. Querétaro,

municipio de Landa de Matamoros, Sierra Gorda, laderas calizas con bosque mesófilo de montaña, 17 Aug de 1996, S. Zamudio &

E. Pérez-Calix 9921 (holotype: IEB!; isotype: IEB!, MEXU).

FIGURE 4. Comparison and visualization of the inverted repeat (IR), small single copy (SSC) and large single copy (LSC) sequences

boundary positions in the 14 North America Magnolia plastomes.

FIGURE 5. Phylogenetic relationships of 14 North America Magnolia accessions inferred from complete plastomes generated by

Bayesian inference approach; values on nodes represent posterior probabilities (PP).

FIGURE 6. Phylogenetic relationships of 14 North America Magnolia accessions inferred from plastid DNA barcodes (matK, rbcL, trnH,

psbA and trnL-F) generated by Bayesian inference approach; values on nodes represent posterior probabilities (PP).

= Magnolia vovidesii A.Vázquez, Domínguez-Yescas & L.Carvajal, Recursos Forest. Occid. México 4: 478. 2013. Type:—MEXICO.

Veracruz. Ixhuacán de los Reyes municipality: 5 km de Coyopolan camino a Ixhuacán, Jul 1988, Vázquez-García 4644 (holotype:

IBUG!; isotypes: F!, MO, WIS).

= Magnolia zotictla A.Sánchez-Gonz., Gut.-Lozano & A.Vázquez Phytotaxa 513: 272. 2021. Type:—MEXICO. Hidalgo: Acaxochitlán

municipality, Zotictla, 0.3 km al SE de San Miguel del Resgate, bosque mesófilo de montaña, 1743 m, 20°13’32.5’’N 98°09’48’’W,

5 May 2021. Gutiérrez-Lozano et al. 10186 (holotype: HGOM; isotypes: ENCB, IBUG, OAX).

ALDABA ET AL.

FIGURE 7. Phylogenetic relationships of 14 North America Magnolia accessions inferred from a group-specific DNA barcode combination

selected from hypervariable regions in Neotropical Magnolia plastomes (ccsA, ndhD, petL, and rpl32) generated by Bayesian inference

approach; values on nodes represent posterior probabilities (PP).

Description:—Trees, 4–35 m high, deciduous. Branches short in pyramidal arrangement. Bark smooth, light brown

with white and dark brown spots. Young branches light green to dark brown, with whitish spots, puberulent with

whitish or yellowish trichomes. Leaves: simple, up to 50 cm long and 30 cm wide, adaxially dark green, glabrous,

abaxially light green to whitish, veins sericeous, base cordate to auriculate, apex acute, margin entire, petioles

cylindrical, sericeous. Stipules deciduous, adnate to the petiole covering up to 60% of it, lanceolate, tomentose. Pedicels

cylindrical, pulverulent to tomentose. Bracts deciduous, ovate, pulverulent at the base. Flowers terminal, solitary,

sepals 3, whitish green, lanceolate, up to 20 cm long and 5 cm wide, adaxially glabrous, abaxially pulverulent, base

truncate, apex acute; petals 6, white, the youngest with an orange or purple spot at base adaxially, elliptic, lanceolate

or ovate, glabrous, coriaceous, up to 25 cm long and 10 cm wide, base obtuse, apex acute. Stamens spirally inserted,

laminar, cymbiform, yellowish, base truncate, apex obtuse, thecae 2, introrse, longitudinal dehiscence. Gynoecium

ovoid, yellowish-white, recurved styles. Peduncles cylindrical, pulverulent. Fruits polyfollicles, ovoid or botuliform,

glabrous, rarely puberulent with yellowish trichomes, 4–15 cm long and up to 10 cm wide, brown, green, orange or

reddish when young, longitudinal dehiscence, follicles beaked. Seeds 1–2 per follicle, obloid to ellipsoid, with funicle,

sarcotesta red.

Distribution and habitat:—Magnolia dealbata is endemic to eastern Mexico, where it is distributed along the

Sierra Madre Oriental mountain range in the states (from north to south) of Nuevo León, Tamaulipas, San Luis Potosí,

Querétaro, Hidalgo, Veracruz, Puebla, and Oaxaca (Fig. 8). It grows in temperate forests: cloud forest, pine forest and

pine-oak forest at elevations of 1000–2300 m.

Common names:—Chirimoya, elosúchil, eloxóchitl, magnolia, ya-nacho yote, yagsa, yolosóchil.

Phenology:—Shedding leaves from October to March, flowering from April to June, and fruiting from June to

September.

Preliminary conservation status:—Although new populations have recently been discovered along the Sierra

Madre Oriental, they consist of only a few individuals. The Extent of Occurrence (EOO) is 77 874.487 km2 and

the Area of Occupancy (AOO) is 268 km2. Hence, following the IUCN criteria (IUCN 2013; IUCN Standards and

Petitions Committee 2024), the proposed assessment category under criterion B is Least Concern (LC). The primary

threats to the species are habitat fragmentation, deforestation and climate change, the most threatened populations are

located in the central part of the SMOr (Hidalgo, Puebla and Veracruz).

FIGURE 8. Map of the known geographic distribution of Magnolia dealbata Zucc. (Magnoliaceae).

Specimens examined:—MEXICO. HIDALGO STATE: Tepehuacán de Guerrero municipality: Chilijapa,

21°0’’19.44’’N 98°51’41.23’’W, 1400 m, 29 Aug. 2015, A. Chávez Cortazar 1015 (XAL); A. Chávez Cortazar

1013 (XAL). Tlanchinol municipality: 3 km al NNE de Chapulhuacán, 1400m, May 1960, F. Sanchez s.n.

(MEXU), Zacualtipán municipality: Vereda entre El Reparo y Zacualtipan, 3 km de la Mojonera, 29 Jun. 1988, A.

Vázquez 4631, M. Cházaro & M. Rosales (MEXU). NUEVO LEÓN STATE: Montemorelos municipality: Ejido

La Trinidad, 25°12’24.28’’N 100°7’13.39’’W, 1526 m, 5 May 2015, A. Chávez Cortazar 990 (XAL). OAXACA

STATE: Huautla de Jiménez municipality: La Providencia, 1500, 19 Apr. 1975, Rzedowski 32842 (MEXU). Ixtlán

de Juárez municipality: Tiltepec, 17°30’48’’N 96°19’29’’W, 1380 m, 25 Apr. 1998, J. García R. 226 (MEXU);

camino a las pueblas de San Juan Yaeé and Tanetze, 17°21’52’’N 96°15’37’’W, 1841 m, 22 Mar. 2016, M. Sundue

et al. 4065 (BRIT, MEXU, VT). San Juan Juquila Vijanos municipality: 5 Km al NE de la desviación a Juquila

Vijanos, hacia Talea, 17°21’28’’N 96°16’19’’W, 1650 m, 11 Jul. 1996, R. Aguilar Santelises 620 (IEB, XAL); La

Cumbre, 17°21’38.39’’N 96°16’27.45’’W, 1924 m, 15 Apr. 2015, A. Chávez Cortazar 715 (XAL). San Juan Yaeé

municipality: San Juan Yaeé, 17°24’28.53’’N 96°16’50.43’’W, 1715 m, 16 Apr. 2015, A. Chávez Cortazar 768. Santa

María Teopoxco municipality: Los Duraznos, 18°8’41.83’’ N 96°57’44.31’’ W, 2187 m, 13 Apr. 2015, A. Chávez

Cortazar 922 (XAL); 39 km de Teotitlan de Flores Magón, por la carretera a Huautla de Jiménez (1-2 km antes de

llegar al Plan de Guadalupe), 16 Apr. 2002, X. Munn-Estrada 2231 y F. Mendoza (MEXU, XAL). Santiago Camotlán

municipality: 500 m al este de Santiago Camotlán, rumbo a Yadoó 17°26’33’’N 96°10’42’’W, 1500 m, 14 Mar. 2013,

M.L. Pérez-Nicolás 109, J. Galicia & O. Quisehualt (IBUG). Talea de Castro municipality: El Faisán, 17°23’8.62’’N

96°15’54.33’’W, 1895 m, 16 Apr. 2015, A. Chávez Cortazar 674 (XAL); Distr. Villa Alta, Talea de Castro a 1.5 km al

N, 25 Mar. 2016, R. Torres Colin 18088, A. Vasco y E. Vasquez Pérez (MEXU). Teotitlán del Camino municipality:

Loma Chapultepec, a 4 km. al S de Huautla de Jiménez, 1750 m, 29 Apr. 1978, M. Sousa S. et al. 9357 (MEXU).

Totontepec Villa De Morelos municipality: Tepitongo, 17°18’00’’N 96°02’00’’W, 1700 m, 10 Mar. 1990, E. Velasco

López 406 (MEXU); Totontepec, 17°15‘N 96°00’W, 1900 m, 9 Mar. 1986, J. Rivera Reyes 0191 & G.J. Martin

(MEXU). Yetzelalag municipality: 1300 m, 5 Mar. 1919, B.P. Riko 4139 (US). Without municipality data: Rincon

(al NE de Totontepec, probablemente San Ildefonso Villa Alta): 1833, Karwinski nd (BR 31083553); Sierra de Ixtlán:

ALDABA ET AL.

21 Apr. 1906, C. Conzatti 1370 (US); 1846, H. Galeotti 4588 (BR 31083577, BR 31083581, BR 31083607, P 1963173,

P 1963172); H. Galeotti 4991 (P 1963175); Karwinski s.n. (M 239896, M 239898, M 239897, M 239899). PUEBLA

STATE: Pahuatlán municipality: Ahila, 20°15’52.83’’ N. 98°10’24.28’’W, 1785 m, 6 Apr. 2015, A. Chávez Cortazar

857 (XAL). Zoquitlán municipality: San Francisco Xitlama, 18°18’32.99’’N 97°1’59.4’’W, 2274 m, 6 Apr 2015,

M. Castañeda-Zárate MCZ-1042 (MEXU). QUERÉTARO STATE: Landa de Matamoros municipality: Joya del

Cedro, 21°14’31.42’’ N, 99° 9’55.58’’W, 1167 m, 31 May 2015, A. Chávez Cortazar 997 (XAL); Joya del Hielo, 17 km

al N de Acatitlán de Zaragoza, 11 Apr. 1989, S. Zamudio & E.Carranza 7197 (MEXU); Joya del Hielo, 21°12’18’’N

99°11’24’’W,1800 m, 17 Aug. 1996, S. Zamudio y E. Pérez C. 9921 (MEXU, XAL); La Lima, 3 km al Noroeste de

La Florida, 21°13’38’’N 99°66’12’’W, 1680 m, 5 May 1989, E. González 540 (MEXU, XAL); Sótano Colorado,

21°14’26.68’’N 99°9’22.44’’W, 1673 m, 4 Oct. 2015, A. Chávez Cortazar 976 (XAL). SAN LUIS POTOSÍ STATE:

Xilitla municipality: José Coronel Castillo, 21°24’21.06’’N 99°4’24.89’’W, 1958 m, 4 May 2016, A. Chávez Cortazar

1211 (XAL); A. Chávez Cortazar 1212 (XAL); A. Chávez Cortazar 1132 (XAL); A. Chávez Cortazar 1209 (XAL).

TAMAULIPAS STATE: Güémez municipality: 1 km al Noreste de Los San Pedro, 1430 m, 8 Jun. 1990, F. González

Medrano 17562, V. Juárez & P. Tenorio (MEXU). Victoria municipality: Rancho El Molino, 1500 m, 1 May 2016,

Leccinum J. Garcia-Morales 5435 (ICTV, MEXU). VERACRUZ STATE: Coatepec municipality: Plan de La Cruz,

carretera antigua Xalapa-Coatepec, 19°30’16’’N 96°56’42’’W, 1358 m, 10 Oct. 2018, F.G. Lorea H. & L.R. Tlaxcalteco

T. 6754 (XAL); Puerto Rico, 19°30’16’’N 96°56’42’’W, 1374 m, 14 May 2018, F.G. Lorea H. & L.R. Tlaxcalteco T.

6706-B (XAL); Plan de la Cruz, 19°30’16’’N 96°56’42’’W, 1374 m, 14 May. 2018, F.G. Lorea H. et al. 6706 (XAL).

Huayacocotla municipality: Helechales, 20°37’N 98°26’W, 1800 m, 5 May 1980, F. Ramírez R. & J. Palma G.

1003 (XAL); Helechales, 12 Aug. 1980, R. Ortega O. 1489 (XAL); El Salto por Agua de la Calabaza, 26 Apr. 1981,

L. Ballesteros & F. Ballesteros 438 (MEXU, XAL); Agua de la Calabaza, 1820 m, 17 May 1980, L. Ballesteros & H.

Morales 214 (XAL); Agua de la Calabaza, 8 Apr. 1980, L. Ballesteros & J. I. Calzada 141 (XAL); Agua de la Calabaza,

23 Mar. 1989, A. Vazquez 4889 (TEX); Agua de la Calabaza, 1820 m, 4 Jun. 1980, L. Ballesteros & H. Morales 260

(MEXU, XAL). Ixhuacan de Los Reyes municipality: Ejido Coyopola, 2 Jun. 1986, L. Gutiérrez Carvajal 13 (XAL);

Ejido Coyopola, 2 Jun. 1986, L. Gutiérrez Carvajal 2 (XAL); 5 km delante de Coyopola en ruta de a pie a Ixhuacán,

1650 m, 28 Aug. 1985, M. Chazaro y P. Padilla 293725 (MEXU, XAL); Coyopolan, 19°22’0.33’’N 97°4’4.40’’W,

1567 m, 11 Apr. 2015, A. Chávez Cortazar 473 (XAL); Coyopola, 19°23’N 97°2’W, 1450 m, 6 Jun. 1984, R. Ortega O

& G. Pattison 2638 (XAL); Coyopolan, 19°22’0.33’’Ν, 97°4’4.40’’ W, 1567 m, 11 Apr. 2015, A. Chávez Cortazar 992

(XAL); Coyopolan, 19°21′58″N; 97°3′11’’W, 1600 m, 18 Jul. 2016, J.A. Guerrero Analco 3 (XAL); Ejido Coyopola,

1500 m, 2 Jun. 1986, L. Gutiérrez Carvajal 12 (XAL); Coyopolan, 19°22’0.33’’N 97°4’4.40’’W, 1567 m, 11 Apr.

2015, A. Chávez Cortazar 994 (XAL); 5 km de Coyopola camino a Ixhuacan, Jul. 1988, A. Vázquez, M. Cházaro & M.

Rosales 4644 (F, IBUG, MEXU); en una cañadita que está entre Coyopola y Tlalchi, 1700 m, 9 Jul. 1984, M. Chazaro

B. s.n. (MEXU); brecha a Coyopola, entrando por la carretera Ixhuacán-Coatepec, 19°21’5.9’’N 97°3’30.8’’W, 1585

m, 3 Sep. 2009, A. Campos V. 6469, S.M. Guzmán T. & A. Troyo B. (MEXU); Coyopolan, 19°21′59″N, 97°4’5’’W,

1570 m, 17 Oct. 2022, D.A. Infante 13 (XAL); Coyopolan, 19°22’0.33’’Ν. 97°4’4.40’’W, 1567 m, 11 Apr 2015, A.

Chávez Cortazar 479 (XAL). Los Reyes municipality: Colonia Bugambilia-Congocotepec (terrenos particulares),

carretera Orizaba-Zongolica (km 32), 18°41’20’’ N, 97°1’18’’ W, 1700 m, 9 Apr. 2000, A. Rincón G. 1413 & C.

Durán E. (MEXU, XAL); Cuacaballo, 18°41’2.77’’ N, 97°1’59.82’’ W, 1736 m, 9 Apr. 2015, A. Chávez Cortazar 874

(XAL). Tequila municipality: Moxala, 18°42’55.42’’ N, 97° 2’15.40’’ W, 1725 m, 10 Apr. 2015, A. Chávez Cortazar

881 (XAL). Tlatetela municipality: Axocuapan, 19°12’18.55’’N 96°59’34.56’’W, 1501 m, 12 Apr. 2015, A. Chávez

Cortazar 915 (XAL). Xalapa municipality: 22 May 1899, J.N. Rose 4316 & W. Hough (US); Along rt. 140, on the

way from Los Pinos to Xalapa, ca. 10 km W of Xalapa, 19°35’28.106’’N 96°57’27.2’’W, 1524 m, J. Wen 17812 (US).

Without municipality data: 23 Mar. 1989, A. Vazquez 4889 (TEX). Without state/locality data: 1975, Paulsen s.n.

(L 1741325).

Notes and taxonomic discussion.:—Magnolia alejandrae, M. nuevoleonensis, M. rzedowskiana, M. vovidesii,

and M. zotictla are synonymized here under Magnolia dealbata Zucc., following the principle of priority according

to the ICN-International Code of Nomenclature (Turland et al. 2018). The different molecular analyses performed

showed that the different taxa form a single entity; no differences were observed in the chloroplast comparisons of the

taxa, and the different phylogenetic analyses of both plastomes and angiosperm plastid DNA barcodes (Figures 5, 6)

showed high levels of support that the taxa form a single entity, and morphospecies are not disjunct in the branches

of the phylogenetic trees but intermingle with each other. Furthermore, the main morphological characters used to

delimit them have no taxonomic weight: the number of carpels is light-dependent and the absence/presence and colour

of the blotch at the base of the petals represents normal development of Magnolia flowers and based on extensive field

sampling of several populations no differences in this colouring are observed; see below for further discussion. Traits

initially identified as unique to some taxa (M. vovidesii, M. zotictla) are better interpreted as polymorphic rather than

diagnostic autapomorphies. This variability supports the hypothesis that they do not represent a distinct taxon, but

rather fall within the variation observed in M. dealbata s.l.

Magnolia dealbata, as a broadly circumscribed species is also supported by geological and climate data. Its

distribution area is nearly entirely restricted to the morphotectonic province of the Sierra Madre Oriental, with a small

portion in the province of the Sierra Madre del Sur sensu Ferrusquía-Villafranca (1993), both areas being separated by

a small area of the Trans-Mexican Volcanic Belt However, until the 1960s its distribution area in southeastern Puebla

and adjacent regions of Veracruz and Oaxaca was even considered to be part of the Sierra Madre Oriental (De Cserna

1961, 1989; Murray 1961). The Sierra Madre Oriental is a nearly continuous system of folded mountain ranges(De

Cserna 1989; Ferrusquía-Villafranca, 1993). Moreover, the species is restricted to the temperate humid and subhumid

climate areas following García (2004), with the northernmost localities of its occurrence area restricted to humid

ravines. Thus, the geological substrate is not a limiting factor for the species but rather characterizes their distribution

according to a particular climate.

There remains the possibility that M. dealbata is a variety of M. macrophylla, as suggested by the specific DNA

barcodes of hypervariable regions. To confirm this hypothesis, it is proposed to include more samples from different

populations of M. ashei and M. macrophylla and compare them phylogenetically with M. dealbata and to make

morphological observations.

Discussion

Magnolia sect. Macrophylla plastome features and variations

Variations of plastomes between different taxa of the Magnolia sect. Macrophylla were minimal (Figures 2, 4),

underscoring the overall conservation of the Magnolia sect. Macrophylla and Magnolia plastome (Guzmán-Díaz et al.

2022; Palmer 1985). Moreover, there were important and noticeable differences between the taxa from the Magnolia

sect. Macrophylla and M. fraseri (Magnolia sect. Auriculata), confirming, in a molecular way, that they are indeed

completely distinct lineages and clades.

In particular, among the taxa of the Magnolia dealbata complex, the two samples that differed most from the

rest were M. vovidesii MA0877A and the one corresponding to M. zotictla. For the former, the variations recorded in

comparison with the other two samples of M. vovidesii could be explained by the fact that this sample comes from

a different population than the rest of M. vovidesii, or that it inhabits a different ecosystem, so these variations are a

response to the habitat or simply related to the individual itself. However, variations in plastomes can be the result of

many other causes, including random mutations or genetic drift (Cao et al. 2024; Chen et al. 2021; Dong et al. 2023;

Du et al. 2022).

Phylogenetic relationships within Magnolia dealbata complex

Phylogenetic hypotheses derived from plastomes and angiosperm plastid DNA barcodes suggest that all taxa in the

Magnolia dealbata complex belong to a single entity (Figures 5, 6). This refutes the initial hypothesis that the different

taxa of the complex would be grouped into two entities in the SMOr (one in the north and one in the south). However,

the topology recovered from the Magnolia-specific plastid DNA barcodes from hypervariable regions suggests a north-

south geographical association (Figure 7). In similar studies, phylogenetic trees based on chloroplast or plastome data

have also resolved relationships more satisfactorily than other sources of molecular evidence (Hu et al. 2023; Li et al.

2022).

In most angiosperms, phylogenetic inconsistencies are commonly recorded between different genes or genomes

as well as among phylogenetic and phylogenomic data (Duan et al. 2023; Jiao et al. 2023; Oliver 2013; Wanke

& Wicke 2023; Zhou et al. 2023; Zuntini et al. 2024), especially between plastome and other molecular datasets

(Giaretta et al. 2022; Hu et al. 2016, 2023; Rokas & Chatzimanolis 2008; Su et al. 2021; Villar et al. 2019). There are

several factors (mainly biological and methodological) that cause conflicts in phylogenies; these include chloroplast

capture, incomplete lineage sorting, hybridisation, introgression in species undergoing rapid radiation and convergent

molecular evolution, sampling error, rate signal, model selection, heterotaxy, and low genetic variability (Cai et al.

2021; Doyle 2022; Hu et al. 2023; Steenwyk et al. 2023; Zhang et al. 2020). The latter has been widely reported for

ALDABA ET AL.

the genus Magnolia (Aldaba Núñez et al. 2021; Budd et al. 2015; Chávez-Cortázar et al. 2021; Guzmán-Díaz et al.

2022; Hernández et al. 2020; Rico & Becerril 2019; Veltjen et al. 2019).

DNA barcodes have been widely used to delimit species complexes and/or problematic angiosperm groups, with

varying results. In some cases, sequences are useful in clarifying the relationships of certain groups (Binh et al. 2018;

Gu et al. 2011; Hu et al. 2023; Su et al. 2021; Zhou et al. 2023), while in others they are of little or no use (Ding et

al. 2022; Du et al. 2011; Starr et al. 2009; Terrones et al. 2022; Zhang et al. 2012). Rather, the current trend is to use

multiple sources of molecular evidence, taking advantage of the fact that next-generation sequencing techniques make

it possible to work with much larger amounts of data at an increasingly lower cost.

Unravelling reproductive organ variations

Phenotypic variations in the reproductive organs of the taxa comprising the Magnolia dealbata species complex used

to distinguish species, are either variations in flowers or in fruits. The former comprises the number of stamens, petal

sizes, and the presence or absence and colour of a purple or yellow spot at the base of the petals, while fruit variations

focus on size, the number of carpels, and whether the latter have a beaked apex (García-Morales et al. 2017; Gutiérrez-

Lozano et al. 2020; Rodríguez-Ramírez et al. 2021; Sánchez-González et al. 2021; Vázquez-García et al. 2012b, 2013,

2015, 2016). Each of these characteristics will be discussed below.

Decoding the colour spectrum: a look at flower variations

The presence and colour of the spot at the base of the petals (either purple or yellowish) have been used as characters of

taxonomic importance to delimit the recently described Mexican taxa of Magnolia sect. Macrophylla (García-Morales

et al. 2017; Sánchez-González et al. 2021; Vázquez-García et al. 2013, 2015, 2016, 2021). However, in the field, it

has been observed that this characteristic is more of a facultative type and may vary. Individuals in bloom have been

recorded with flowers that may or may not have the blotch, i.e., they have flowers with the blotch, others with a less

marked, barely perceptible blotch, and even other flowers that are completely white, all occurring on the same tree

(pers. obs.).

In addition, a similar phenological phenomenon has been observed in the US taxa of the section: M. ashei and M.

macrophylla (Chafin 2000; Gilman & Watson 1994; Meyer 1993; Pattison 1985), as well as in other Magnolia taxa

from Magnolia sect. Talauma (Aldaba Núñez 2020), which are also distributed in the SMOr (specifically M. mexicana

DC.). On the one hand, both M. ashei and M. macrophylla are described with only the innermost petals as purple, while

the outer petals are greenish, but it has been observed that the outermost petals also exhibit the purple stain and are the

last to lose it so that in the most mature flowers both the inner and outer petals are completely white (pers. obs.). There

are also literature sources reporting entire populations of M. macrophylla in Alabama and Mississippi with all-white

flowers (Treseder & Blamey 1981). On the other hand, in M. mexicana, the youngest flowers have a completely purple

colouring, which fades as they open and continue to mature so that the purple hue is reduced until it becomes a spot

at the base of the petals that finally disappears completely so that the most mature flowers are white and devoid of the

purple spot (pers. obs.).

Based on the above, it could be deduced that the presence of an orange stain instead of a purple blotch in the taxa

of the Magnolia sect. Macrophylla is more likely to be a consequence of such a fading of the colouring from purple to

white; this pattern has also been seen in other angiosperms (Bar-Akiva et al. 2010; Rezende et al. 2020; Vaknin et al.

2005). Furthermore, no ecological correlation (such as differences in vegetation types, soil, or climate) or geographical

correlation (e.g. distribution in altitudinal ranges or north-south longitude) has been found that could explain the

presence of such a patch. That is, one would expect that taxa from the North would present such a spot, while those

from the South would not or vice versa (and perhaps those from the centre would be the ones to present the orange

colouration, thus exhibiting a spectrum of gradation in petal shades). However, this is not the case, it is present in an

interspersed manner in the taxa regardless of their distribution. The same is true when the altitudinal range of the taxa

is taken into account, which does not influence the presence or absence of the spot at the base of the petals.

However, to understand this phenomenon in depth, phenological and phytochemical studies are needed to elucidate

the development and function of these purple shades and their changes during the process of flower maturation. Colour

change in angiosperm flowers can be due to various factors, such as soil pH, senescence, sun exposure, or even in

response to pollinators (Casper & La Pine 1984; Cruzan et al. 1988; Del Valle et al. 2019; Delph & Lively 1989; Gori

1989; Ida & Kudo 2003; Jones & Cruzan 1999; Kudo et al. 2007; Li et al. 2019; Luo et al. 2017; Martínez-Harms et

al. 2022; Narbona et al. 2021; Oberrath & Böhning-Gaese 1999; Odell et al. 1999; Ram & Mathur 1984; Ruxton &

Schaefer 2016; Teppabut et al. 2018; Weiss 1991; Weiss & Lamont 1997; Zhang et al. 2023). In the particular case

of magnolias, the colour change may be associated more with senescence, as has been documented in other families

(Brito et al. 2015; Teppabut et al. 2018; Weiss 1995).

Fruitful insights: exploring fruit variations

Characters related to fruit shape and size have also been used to delimit the different taxa that make up the species

complex studied. However, based on the detailed observations of herbarium specimens, it was noted that there is a

marked intraspecific morphological variation in both shape and size; therefore, these characteristics are not of taxonomic

importance. Particularly noteworthy is the case of M. alejandrae, a taxon which, according to the description, has the

smallest fruits of all the taxa in both the complex and the section (4–7 cm × 3–4.5 cm; García-Morales et al. 2017),

but specimens with larger fruits were observed, whose measurements overlap with the other taxa of the complex, thus

making taxonomic delimitation difficult. It is precisely this situation, which also occurs in the other synonymised taxa,

complemented by molecular data, both from previously studied microsatellites (Chávez-Cortázar et al. 2021) and from

the phylogenomic data generated in the present study, that has led to the taxonomic decisions presented.

Another of the main morphological characteristics traditionally used to delimit Magnolia species, especially in the

Neotropical region, is the number of carpels (Cruz-Durán et al. 2014; García-Morales et al. 2017; Lozano-Contreras

1994; Vázquez-García 1994; Vázquez-García et al. 2013, 2015, 2021). However, when statistical analyses based on

a large sample of fruits have been carried out, it has been found that carpel numbers overlap among taxa (Aldaba

Núñez 2020). Furthermore, it has been shown in model organisms that this trait is genetically related and regulated by

light (Reymond et al. 2012). In addition, some recent studies on the evolutionary processes of carpels in angiosperms

have concluded that these processes are quite complex due to their diversity in shape and arrangement; their ontogeny

is subject to a large number of developmental processes, which are well documented for Arabidopsis thaliana (L.)

Heynhold (1842: 538), but not for non-model organisms (Liu et al. 2000, 2022; Pfannebecker et al. 2016; Reyes-

Olalde et al. 2023; Rivarola 2020; Sattler 2024). In this species complex, it has been observed that fruits from the

same tree that were more exposed to sunlight had a higher number of carpels than those that were more shaded (pers.

obs.).

Shedding light on sterile organ variations

Once the morphological variations of the reproductive organs have been analysed, the variations of the sterile organs

remain to be dealt with. The main characteristic of this type, that has been proposed as taxonomically important for

delimiting taxa within the Magnolia dealbata complex, is leaf size (García-Morales et al. 2017; Gutiérrez-Lozano et

al. 2020; Sánchez-González et al. 2021; Vázquez-García et al. 2013, 2015, 2016).

Magnolia leaves exhibit considerable variation in size and shape (Gutiérrez-Lozano et al. 2020; Rodríguez-Ramírez

et al. 2021). Morphological observations did not reveal any pattern that would allow the different morphospecies of the

complex to be separated by leaf size or shape. It was also observed that other traits, such as leaf pubescence, showed

a wide phenotypic variation, especially in individuals that had been identified as M. vovidesii, where specimens from

the same locality can be either glabrous or pubescent.

Other deciduous plant organs, such as stipules and bracts, are less conspicuous and thus underrepresented in

collections, often remaining overlooked (Aldaba Núñez 2020). These organs exhibit relatively stable and conserved

morphology, which could be significant for species delimitation, particularly concerning the type of pubescence.

However, it was also observed that these structures did not prove useful in delineating species within the complex.

Finally, some discrepancies were observed in the morphology of the studied taxa. For instance, in M. mixteca,

although molecular samples were not included in this study, morphological observations were made. The taxon’s

description mentions that the stipule has sericeous pubescence (Vázquez-García et al. 2021). However, specimens

from the type locality were observed with a completely glabrous stipule. In principle, this suggests that it indeed

may be a distinct taxon. However, additional field expeditions are required to gather more specimens and make a

determination.

Overall, the traits that exhibit the most intraspecific variation are the ones used to define the new taxa of the

Magnolia dealbata complex (number of carpels, number of stamens, length of leaves (Gutiérrez-Lozano et al. 2020).

It may be tempting to consider these variations as a result of significant phenotypic plasticity, but further study is

required. Magnolia morphology is more intricate than it appears, and this phenomenon has only recently been explored

ALDABA ET AL.

in depth (Aldaba Núñez et al. 2024; Gutiérrez-Lozano et al. 2020; Lozano-Contreras 1994; Rodríguez-Ramírez et al.

2021). Additionally, the morphological characteristics used to define certain sections may not apply to other sections.

As part of the morphological observations, characters previously proposed as diagnostic autapomorphies for

certain taxa—such as the indumentum on peduncles and petioles in M. zotictla—were re-evaluated. Field and

herbarium studies revealed considerable variability in these characters, even between individuals within the same

population. For example, peduncle pubescence ranged from pulverulent to sericeous, while petiole indumentum varied

from glabrous to sericeous. This evidence suggests that these characters are better interpreted as polymorphic rather

than autapomorphic, highlighting their inadequacy for diagnosing M. zotictla as a distinct species. Interestingly, M.

zotictla stands out within the Magnolia dealbata complex as the morphospecies with the most restricted distribution

and the smallest known population size (Sánchez-González et al. 2021). These findings underscore the phenotypic

plasticity and morphological variation present within each morphospecies of the Magnolia dealbata complex

Taxonomic limits within the Magnolia dealbata complex

In the last two decades, around 80 new Magnolia species have been described in the Neotropical region; consequently,

nearly half of the world’s Magnolia species are now found in the Neotropics. These newly segregated taxa often have

narrow distributions and are sometimes microendemic. (i.e., taxa segregated from M. schiedeana and M. mexicana

(Vázquez-García et al. 2012b, 2013). However, the case of M. dealbata sensu lato may be an exception to this rule.

Although the distribution of the closest taxon (M. macrophylla) is quite wide compared to other species in other sections

(García-Morales et al. 2017; Gilman & Watson 1994; Meyer 1993), it is important to note that this is not always the

case. In general, Magnolia species from temperate clades tend to have larger distributions than those from tropical

habitat sections (pers. obs.), which could also be since there are more studies in temperate zones than in tropical ones,

which are more difficult to access; it could also be due to the fact that temperate climates are wider and more stable.

In the tropics, climatic conditions can change dramatically within a few kilometres, so the plant has a limited range

(Sentinella et al. 2020). However, this statement needs to be investigated, as several factors explain the distribution

and diversification of angiosperms (Gehrke 2018; Pennington et al. 2009; Tietje et al. 2022). The deciduous leaves of

Magnolia sect. Macrophylla taxa may contribute to their broader geographical distribution, suggesting adaptability to

diverse habitats. However, the distribution of M. dealbata s.l. across various forest types in the SMOr indicates that

factors beyond leaf type influence their occurrence. Interestingly, despite their wider distribution, deciduous groups of

Magnolia in both continents have lower species richness compared to evergreen groups, hinting at complex underlying

factors that govern species diversity.

Furthermore, the minimal variation in plastome structure and arrangement suggests a single identity with multiple

populations comprising Magnolia dealbata complex taxa and potentially the two US varieties of M. macrophylla.

However, from a broader perspective, both US and Mexican taxa could belong to the same entity: M. macrophylla,

with Mexican taxa forming the variety M. macrophylla var. dealbata (Zucc.) D.L: Johnson (1989: 55), as previously

proposed (Johnson 1989). Additional samples of M. macrophylla from the USA are needed to confirm this hypothesis.

When comparing plastomes of different taxa within a species complex, differences and variations in plastomes are

usually evident and consistent with the results of phylogenetic hypotheses (Cao et al. 2024; Hu et al. 2023; Li et al.

2022). Although the various studies of morphological and molecular variation conclude that the different taxa that

make up the Magnolia dealbata complex are indeed differentiated species, the results obtained here suggest otherwise.

On the one hand, morphological variation is based on very few characters from a larger matrix, so most morphological

characters support the hypothesis that the taxa form a single entity with specific morphological variations (Gutiérrez-

Lozano et al. 2020; Rodríguez-Ramírez et al. 2021). On the other hand, the most recent molecular results from

microsatellites do indeed show that individuals of the different taxa intermingle, and no real differentiation is observed

(López-Ramírez et al. 2024), as previously reported (Chávez-Cortázar et al. 2021). This could be a matter of scale: if

sampling is extended to include most of the taxa in the complex, little variation is observed, but if it is focused on a

smaller number of taxa and populations, more differentiation is observed, and genetic groups become more apparent.

The species complex has a discontinuous distribution along the SMOr, the gaps can be explained conclusively,

as it is highly probable that M. dealbata can be found in these areas, as several of them have a similar climate and

vegetation type suitable for M. dealbata. From north to south, the gap between the populations of ‘M. nuevoleonensis’

in Nuevo León and those of ‘M. alejandrae’ in Tamaulipas is explained by the fact that this area is difficult to access

and has been little studied (Martínez Salas, pers. comm.). Next, the gap between the populations of ‘M. alejandrae’

and those of ‘M. rzedowskiana’ in San Luis Potosí and ‘M. zotictla’ in Hidalgo and Puebla is due to the lower foothills

and drier climate, which limits their distribution to the Huasteca, a more humid area that represents the northern limit

of the high forests; however, around Ciudad del Maiz, San Luis Potosí in the middle of the gap there are reports of

montane cloud forest (Martínez Salas, pers. comm.). Although generic differentiation between both morphospecies has

been reported, it was not significant (Chávez-Cortázar et al. 2021). Subsequently, the gap between the populations of

‘M. rzedowskiana’–‘M. zotictla’ and ‘M. vovidesii in Veracruz is explained by the steep slopes in the Misantla area,

(Martínez-Salas, pers. comm.). Finally, the gap between the populations of ‘M. vovidesii’ and M. dealbata s.s. is since

there are several natural communal zones in the area, to which the local people do not allow access, which is another

little explored area, but this time due to social factors rather than natural factors (Martínez Salas, pers. comm.).

Despite these gaps, it must be considered that magnolias are pollinated by beetles and dispersed by birds, organisms

that can easily evade the steep slopes of the SMOr (Gottsberger et al. 2012; Gutiérrez-Zúniga 2018; Sun et al. 2023;

Thien 1974; Wang et al. 2014; Werle 2002). Furthermore, magnolias from other sections (i.e., M. tamaulipana from

sect. Magnolia in Reserva de la Biósfera El Cielo, Tamaulipas and M. mexicana from Magnolia sect. Talauma in the

Sierra Norte de Puebla, Puebla) can be found in these areas, which reinforces the fact that these gaps are due to limited

collection efforts and that M. dealbata could inhabit these areas and maintained historical gene flow throughout the

SMOr, as showed in a previous study (Chávez-Cortázar et al. 2021).

Magnolia mixteca is another species of the Magnolia sect. Macrophylla but it occurs in a different mountain

range, the SMS in Oaxaca state, so it was not included as the study focused on the SMOr species. This species was

described “de novo”, rather than being segregated populations from M. dealbata as happened with the SMOr taxa

(Vázquez-García et al. 2021).

Implications for conservation

During our field expeditions, we observed that magnolias are integral components of the primary vegetation (particularly

in cloud forests) and are particularly vulnerable to environmental disturbances. We especially found individuals away

from forest margins, where the species composition indicated less disturbance, suggesting that these species may prefer

more secluded, undisturbed habitats, as also reported in the literature (Chávez-Cortázar et al. 2021; López-Ramírez et

al. 2024) and observed in other sections (Aldaba Núñez et al. 2021; Budd et al. 2015; Hernández et al. 2020; Sánchez-

Velásquez et al. 2016). Furthermore, due to their medicinal properties, these plants are often indiscriminately collected

for commercial purposes, especially their flowers, preventing fruiting and reproduction. Their distinctive growth

habit makes them easy to identify in the field, but unfortunately, this also makes them an easy target for collectors.

Historical and current literature note that their flowers are highly aromatic (Argueta-Villamar 2009; Hernández 1959;

Hernández-Cerda 1988; Meyer 1993; Mociño & Sessé 2010), which not only aids in their location but also increases

their collection appeal. Despite this, there are places, such as Xalapa, Veracruz, where people are more protective of the

species and use it for ornamental purposes, and where the trade is more local, without intensive exploitation.

According to the IUCN Red List (IUCN 2013, 2021; IUCN Standards and Petitions Committee 2024), five of

the taxa that comprise the complex are classified as ‘Endangered’ (Akande & Yobal 2020; Rivers 2015b, 2016a; b;

Vasquez-Garcia et al. 2023), with M. zotictla being the most threatened with a ‘CR’ category (Critically Endangered;

(Caeaeun Her & Sanchez Gonzalez 2023), while only M. dealbata s.s. is classified as ‘NT’ (Near Threatened; (Rivers

2015a). However, we propose a new provisional conservation status for M. dealbata as Least Concern (LC). Magnolia

dealbata occupies an extensive EOO covering the entire range of the SMOr, although the AOO value falls into the EN

category. It is mainly found in cloud forests, the most threatened habitat in Mexico, and the one that has suffered the

greatest area loss (Castillo-Hernández & Flores-Olvera 2017; Domínguez-Yescas et al. 2020; Gual-Díaz & Rendón-

Correa 2017; Ramírez-Bamonde et al. 2005; Williams-Linera et al. 2016a). Although new small populations have been

discovered, in the central part of the SMOr, in the southern SMOr larger populations can be found, sometimes forming

forests of up to 250 individuals, especially in Oaxaca (Galindo 2010; García Padilla et al. 2022; Rodríguez-Robayo

& Merino-Pérez 2018). The species is found in several federal, state and communal natural areas in Mexico, which

reduces conservation concerns. This is particularly the case in Oaxaca, the state with the highest number of communal

natural areas and where, due to the uses and customs of its inhabitants, it is one of the few places in Mexico and the

world where forest cover has increased in the last 50 years (Martínez Salas, pers. comm.).

In addition, further exploration and work on population genetics is suggested, especially in the northern and

southern parts of the SMOr, considering that all populations belong to the same species, as exposed here, to develop

appropriate conservation strategies and propose conservation units. As well as including M. mixteca in conservation

genetics studies, this taxon is under the Endangered (EN) category according to the Red List (IUCN 2013, 2021;

Vasquez-Garcia et al. 2023), so more considerable conservation efforts are needed.

ALDABA ET AL.

Conclusion

In this study, comparative analyses of the complete chloroplast of a species complex of Magnolia were performed.

They can be a source of information on species relationships and taxonomic changes in taxa.

Several sources of molecular evidence were used: plastomes, angiosperm plastid DNA barcodes, and Magnolia-

specific plastid DNA barcodes obtained from hypervariable plastid regions. The first two sources of evidence suggest

that the Mexican taxa of the Magnolia dealbata complex studied form a single entity.

Traditional morphological traits used to distinguish Magnolia species are no longer sufficient due to the increased

description of new species and the discovery of new populations. Specifically, the number of carpels and the presence

or absence of a colour spot at the base of the petals, traditionally used to delimit species, have been found to be less

reliable. This underscores the need for a reassessment of the morphological characters used in Magnolia taxonomy

and the proposal of new morphological characteristics. The development of more reliable morphological markers for

species delimitation will have important implications for studies on the medicinal uses and conservation efforts of

these taxa. Accurate species identification is essential for understanding their medicinal properties. It is also suggested

that historical, geological, and ecological factors be taken into account when considering separating populations and

elevating them to species rank. Although there may be gaps in the distribution of populations of a species, these can be

explained when considering the vegetation, geology, geomorphology, climate, and previous collections in the region.

Further research on this species complex is suggested. The present study represents a preliminary advance in the

delimitation of Mexican Magnolia sect. Macrophylla species based on molecular and morphological data. However,

further morphometric and statistical studies can be carried out to provide some quantitative support to the taxonomic

analysis, which is more qualitative in nature.

Finally, based on the results, an update of the conservation status of M. dealbata as Least Concern (LC) is proposed

so that it would no longer be in any risk category according to the IUCN Red List guidelines.

Acknowledgements

The authors thank the authorities in Mexico for the collection permits (SGPA/DGGFS/712/1643/13 and SGPA/

DGGFS/712/1063/18). We are grateful to the following colleagues who supported us with lab or fieldwork, providing

samples, logistics and/or obtaining the necessary permits: Pieter Asselman, Angélica Chávez Cortázar, Isabel Larridon,

Martín Mata Rosas, and Emily Veltjen. This work was supported by the Fondation Franklinia 2019-003; the Arboretum

Wespelaar; the King Leopold III Fund for Nature Exploration and Conservation; the Instituto de Ecología, A.C. and

the Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAHCYT scholarships 916022 and 848616 to FA

and SG).

Author contributions

Conceptualization: FAAN. Writing–original draft: FAAN. Writing–review and editing: EMMS, FAAN, MSS, SK.

Data curation: EMMS, FAAN, SGD, SK, SP. Formal analysis: FAAN, SGD. Investigation: FAAN. Methodology:

FAAN, SGD. Project administration: FAAN, MSS. Visualization: FAAN. Resources: SGD, SK, SP. Software: SGD.

Validation: EMMS, MSS. SGD. Supervision: EMMS, MSS. Funding acquisition: MSS.

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Supplementary Materials. The following supporting information can be downloaded at the DOI landing page

of this paper:

Supplementary file 1. Sequence identity plot produced by Shuffle-LAGAN alignment in mVista comparing 13 North

American Magnolia taxa using Magnolia fraseri Walter as the reference. The x-axis represents the base sequence of the

alignment, and the y-axis represents per cent identity (50–100%). Grey arrows represent genes with their orientation.

Pink areas represent conserved non-coding sequences (CNS). Blue areas represent exons.

Supplementary figure 1. Phylogenetic relationships of 14 North America Magnolia accessions inferred from complete

plastomes generated by maximum Likelihood approach; values on nodes represent bootstrap support percentages

(BP).

Supplementary figure 2. Phylogenetic relationships of 14 North America Magnolia accessions inferred from plastid

DNA barcodes (matK, rbcL, trnH, psbA and trnL-F) generated by maximum Likelihood approach; values on nodes

represent bootstrap support percentages (BP).

Supplementary figure 3. Phylogenetic relationships of 14 North America Magnolia accessions inferred from a group-

specific DNA barcode combination selected from hypervariable regions in Neotropical Magnolia plastomes (ccsA,

ndhD, petL, and rpl32) generated by maximum Likelihood approach; values on nodes represent bootstrap support

percentages (BP).

ResearchGate has not been able to resolve any citations for this publication.

Structure, diversity, and genetic delimitation of two Magnolia species endemic to eastern Mexico

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In Mexico, Magnolia species of the section Macrophylla have been segregated based on phenotypic characteristics. However, the wide environmental variation throughout their distribution area in the eastern region of the country could be the main cause of the high degree of morphological variation among the taxa of this section. Therefore, it is necessary to carry out studies at the genetic level to confirm the taxonomic identity of these species. In the present study, the diversity and genetic structure of the populations of Magnolia rzedowskiana and M. zotictla were estimated using simple sequence repeat markers. We evaluated whether the taxonomic delimitation as independent species is consistent with their degree of genetic differentiation, comparing three regions of chloroplast DNA: trnH-psbA, ORF350, and rpl32-trnL. The DNA was obtained from four populations of the two species, which are distributed in the states of Hidalgo, Querétaro and San Luis Potosí. The populations presented intermediate–high genetic diversity (He = 0.52–0.60), limited gene flow (Nm = 0.62) and high genetic differentiation (Fst = 0.288), which may be related to their low density and strong geographic isolation, caused by the high degree of fragmentation of their habitat. The phylogenetic analysis and the genetic differentiation values indicate that each of the populations of the two Magnolia species analyzed behaves as an independent evolutionary unit, so the taxonomic delimitation of the three M. rzedowskiana populations must be reconsidered. The results obtained could be very useful for implementing management and conservation strategies for these populations in the short term.

Floral traits and their connection with Pollinators and Climate

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Mar 2024
ANN BOT-LONDON

Background and Aims Floral characteristics vary significantly among plant species, and multiple underlying factors govern this diversity. Although it is widely known that spatial variation in pollinator groups can exert selection on floral traits, the relative contribution of pollinators and climate to the variation of floral traits across large geographic areas remains a little-studied area. Besides furthering our conceptual understanding of these processes, gaining insight into the topic is also of conservation relevance: understanding how climate may drive floral traits variation can serve to protect plant-pollinator interactions under global change conditions. Methods We used Rhododendron as a model system and collected floral traits (corolla length, nectar volume and concentrations), floral visitors, and climatic data on 21 Rhododendron species across two continents (North America-Appalachians and Asia-Himalaya). Based on this we quantified the influence of climate and pollinators to floral traits using phylogeny-informed analyses. Key results Our results indicate that there is substantial variation in pollinators and morphological traits across Rhododendron species and continents. We came across four pollinator groups: birds, bees, butterflies, and flies. Asian species were commonly visited by birds, bees, and flies, while bees and butterflies were the most common visitors of North American species. The visitor identity explained nectar trait variation, with flowers visited by birds presenting higher volumes of dilute nectar and those visited by insects producing concentrated nectar. Nectar concentration and corolla length exhibited a strong phylogenetic signal across the analysed set of species. We also found that nectar trait variation in the Himalaya could also be explained by climate, which presented significant interactions with pollinator identity. Conclusions Our results indicate that both pollinators and climate contribute and interact to drive nectar trait variation, suggesting that both can affect pollination interactions and floral (and plant) evolution individually and interacting with each other.

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Rolf Sattler

Since the 19th century, we have had countless debates, sometimes acrimonious, about the nature of the gynoecium. A pivotal question has been whether all angiosperms possess carpels or if some or all angiosperms are acarpellate. We can resolve these debates if we do not define the carpel as a closed megasporophyll but simply as an appendage that encloses the placenta or a single ovule. This redefinition may, however, lead to confusion because often it may not be clear whether the traditional (classical) definition of the carpel or the redefinition is implied. Therefore, a topographic approach is proposed that is compatible with the redefinition. According to this approach, gynoecia comprise one or more gynoecial appendages and placentas or single ovules that may be formed in different positions. Heterotopy refers to these different positions. In the context of evo-devo, which explores evolutionary changes in development, morpho evo-devo delves into spatial shifts of the placentas and ovules leading to heterotopy. Furthermore, it considers shifts in timing (heterochrony) and other processes leading to heteromorphy. Recognizing spatial shifting of the placentas or a single ovule and other evolutionary processes opens up new vistas in the search for the ancestor(s) of angiosperms and their gynoecia.

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Genome-scale data and the development of novel statistical phylogenetic approaches have greatly aided the reconstruction of a broad sketch of the tree of life and resolved many of its branches. However, incongruence - the inference of conflicting evolutionary histories - remains pervasive in phylogenomic data, hampering our ability to reconstruct and interpret the tree of life. Biological factors, such as incomplete lineage sorting, horizontal gene transfer, hybridization, introgression, recombination and convergent molecular evolution, can lead to gene phylogenies that differ from the species tree. In addition, analytical factors, including stochastic, systematic and treatment errors, can drive incongruence. Here, we review these factors, discuss methodological advances to identify and handle incongruence, and highlight avenues for future research.

Phylogenomics and plastome evolution of Indigofera (Fabaceae)

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Jun 2023

Introduction Indigofera L. is the third largest genus in Fabaceae and includes economically important species that are used for indigo dye-producing, medicinal, ornamental, and soil and water conservation. The genus is taxonomically difficult due to the high level of overlap in morphological characters of interspecies, fewer reliability states for classification, and extensive adaptive evolution. Previous characteristic-based taxonomy and nuclear ITS-based phylogenies have contributed to our understanding of Indigofera taxonomy and evolution. However, the lack of chloroplast genomic resources limits our comprehensive understanding of the phylogenetic relationships and evolutionary processes of Indigofera. Methods Here, we newly assembled 18 chloroplast genomes of Indigofera. We performed a series of analyses of genome structure, nucleotide diversity, phylogenetic analysis, species pairwise Ka/Ks ratios, and positive selection analysis by combining with allied species in Papilionoideae. Results and discussion The chloroplast genomes of Indigofera exhibited highly conserved structures and ranged in size from 157,918 to 160,040 bp, containing 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Thirteen highly variable regions were identified, of which trnK-rbcL, ndhF-trnL, and ycf1 were considered as candidate DNA barcodes for species identification of Indigofera. Phylogenetic analysis using maximum likelihood (ML) and Bayesian inference (BI) methods based on complete chloroplast genome and protein-coding genes (PCGs) generated a well-resolved phylogeny of Indigofera and allied species. Indigofera monophyly was strongly supported, and four monophyletic lineages (i.e., the Pantropical, East Asian, Tethyan, and Palaeotropical clades) were resolved within the genus. The species pairwise Ka/Ks ratios showed values lower than 1, and 13 genes with significant posterior probabilities for codon sites were identified in the positive selection analysis using the branch-site model, eight of which were associated with photosynthesis. Positive selection of accD suggested that Indigofera species have experienced adaptive evolution to selection pressures imposed by their herbivores and pathogens. Our study provided insight into the structural variation of chloroplast genomes, phylogenetic relationships, and adaptive evolution in Indigofera. These results will facilitate future studies on species identification, interspecific and intraspecific delimitation, adaptive evolution, and the phylogenetic relationships of the genus Indigofera.

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Myricaria plants are widely distributed in Eurasia and are helpful for windbreak and embankment protection. Current molecular evidence has led to controversy regarding species boundaries within the Myricaria genus and interspecific phylogenetic relationships between three specific species— M. bracteata , M. paniculata and M. squamosa —which have remained unresolved. This study treated these three unresolved taxa as a species complex, named the M. squamosa complex. The genome skimming approach was used to determine 35 complete plastome sequences and nuclear ribosomal DNA sequences for the said complex and other closely related species, followed by de novo assembly. Comparative analyses were conducted across Myricaria to identify the genome size, gene content, repeat type and number, SSR (simple sequence repeat) abundance, and codon usage bias of chloroplast genomes. Tree-based species delimitation results indicated that M. bracteata , M. paniculata and M. squamosa could not be distinguished and formed two monophyletic lineages (P1 and P2) that were clustered together. Compared to plastome-based species delimitation, the standard nuclear DNA barcode had the lowest species resolution, and the standard chloroplast DNA barcode and group-specific barcodes delimitated a maximum of four out of the five species. Plastid phylogenomics analyses indicated that the monophyletic M. squamosa complex is comprised of two evolutionarily significant units: one in the western Tarim Basin and the other in the eastern Qinghai-Tibet Plateau. This finding contradicts previous species discrimination and promotes the urgent need for taxonomic revision of the threatened genus Myricaria . Dense sampling and plastid genomes will be essential in this effort. The super-barcodes and specific barcode candidates outlined in this study will aid in further studies of evolutionary history.

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