Materials and Methods
The research objects –
As an ideal stress-test basis for evaluating repeatability of
morphometric studies in insect systematic research, we selected ten
specimens each of a cryptic species pair, Nesomyrmex devius(Csősz & Fisher, 2016) and N. hirtellus (Csősz & Fisher, 2016),
for a total of twenty ant specimens. Every trait under observation shows
overlapping ranges (Seifert 2009); thus, these species can be classified
in multivariate fashion only. Today, cryptic species pairs are
considered the most difficult cases and pose extraordinary challenges to
systematic biology.
The material is deposited in the California Academy of Sciences, San
Francisco, California, U.S.A. The full list of material morphometrically
examined in this work is listed in Supplementary Table S1 (available on
Dryad athttps://doi.org/10.5061/dryad.q83bk3jfq).
Because two specimens suffered a certain degree of damage during the
projects due to consecutive postal shipments, making the subsequent
gaugers unable to measure them, final analyses were done on only 18
individuals. The ant specimens used in this study comply with the
regulations for export and exchange of research samples outlined in the
Convention on Biological Diversity and the Convention on International
Trade in Endangered Species of Wild Fauna and Flora. For field work
conducted in Madagascar, permits to research, collect, and export ants
were obtained from the Ministry of Environment and Forest as part of an
ongoing collaboration between the California Academy of Sciences and the
Ministry of Environment and Forest, Madagascar National Parks and Parc
Botanique et Zoologique de Tsimbazaza (Approval Numbers: N°
0142N/EA03/MG02, N° 340N-EV10/MG04, N° 69 du 07/04/06, N°
065N-EA05/MG11, N° 047N-EA05/MG11, N° 083N-A03/MG05, N° 206
MINENVEF/SG/DGEF/DPB/SCBLF, N° 0324N/EA12/MG03, N° 100
l\fEF/SG/DGEF/DADF/SCBF, N° 0379N/EA11/MG02, N°
200N/EA05/MG02). Authorization for export was provided by the Director
of Natural Resources.
Gaugers –
We addressed the question of whether or not the morphometric
measurements performed by eleven gaugers (“measurers”) could be
considered repeatable based on statistical thresholds. Eleven volunteers
from three continents and six countries, who all have different levels
of taxonomic training and skill, were asked to perform a pair of
measurements on the same set of ant specimens with their own equipment.
Eight of the volunteers are myrmecologists and three are
non-myrmecologists (two are wasp specialists and one is a
dipterologist). The wide range of the observers’ morphometric skills and
the different levels of laboratory facilities and equipment, especially
the types of microscopes used, provided an overview of morphometric
reproducibility as it works in daily practice. Data belonging to gaugers
appear anonymously in this paper, but in order to provide the most
important information regarding their skills and their equipment’s
quality, gaugers are coded in triad format as follows: expertise in
field, estimated total number of specimens measured in their career, and
the maximum magnification of the microscope used in the present study
separated by underscores (e.g. MYRM_9000_100x).
The morphometric character recording protocol
–
Gaugers were asked to measure 21 continuous morphometric characters in
each specimen twice in order to collect data for testing both
intra-gauger error, equivalent to repeatability, and inter-gauger error
rate, equivalent to reproducibility. Every gauger was provided the same
measurement protocol, including visual and verbatim trait definitions to
follow (Fig. 2 and Table 1). The protocol was assembled based on an
existing set of characters used in published papers (Seifert, 2006,
2018; Csősz & Fisher, 2016; Schlick-Steiner et al., 2006; Wagner et
al., 2017). In the current work, we addressed the question of to what
extent random and systematic errors affect the rate of reproducibility.
Therefore, all gaugers were encouraged to eliminate extraordinary
differences due to gross error (occurring due to misreading, mistyping
or erroneously set magnification) by comparing the values of the
repeated observations.
Table 1. Verbatim trait definitions for morphometric character
recording.